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Endurance exercise training improves exercise capacity as well as skeletal muscle and whole body metabolism, which are hallmarks of high quality-of-life and healthy aging. However, its mechanisms are not yet fully understood. Exercise-induced mitophagy has emerged as an important step in mitochondrial remodeling. Unc-51-like autophagy-activating kinase 1, ULK1, specifically its activation by phosphorylation at serine 555, was discovered as an autophagy driver and to be important for energetic stress-induced mitophagy in skeletal muscle, making it a potential mediator of the beneficial effects of exercise on mitochondrial remodeling. Here, we used CRISPR/Cas9-mediated gene editing and generated knock-in mice with a serine-to-alanine mutation of Ulk1 on serine 555. We now report that these mice displayed normal endurance capacity and cardiac function at baseline with a mild impairment in energy metabolism as indicated by an accelerated increase of respiratory exchange ratio (RER) during acute exercise stress; however, this was completely corrected by 8 wk of voluntary running. Ulk1-S555A mice also retained the exercise-mediated improvements in exercise capacity and metabolic flux. We conclude that Ulk1 phosphorylation at S555 is not required for exercise-mediated improvements of exercise and metabolic capacity in healthy mice. NEW & NOTEWORTHY: We have used CRISPR/Cas9-mediated gene editing to generate Ulk1-S555A knock-in mice to show that loss of phosphorylation of Ulk1 at S555 blunted exercise-induced mitophagy and mildly impairs energy metabolism during exercise in healthy mice. However, the knock-in mice retained exercise training-mediated improvements of endurance capacity and energy metabolism during exercise. These findings suggest that exercise-induced mitophagy through Ulk1 activation is not required for the metabolic adaptation and improved exercise capacity in young, healthy mice. [ABSTRACT FROM AUTHOR]

We have reported previously that during hypoxia exposure, the expression of mature miR-17~92 was first upregulated and then downregulated in pulmonary artery smooth muscle cells (PASMC) and in mouse lungs in vitro and in vivo. Here, we investigated the mechanisms regulating this biphasic expression of miR-17~92 in PASMC in hypoxia. We measured the level of primary miR17~92 in PASMC during hypoxia exposure and found that short-term hypoxia exposure (3% O2, 6 h) induced the level of primary miR-17~92, whereas long-term hypoxia exposure (3% O2, 24 h) decreased its level, suggesting a biphasic regulation of miR17~92 expression at the transcriptional level. We found that short-term hypoxia-induced upregulation of miR-17~92 was hypoxiainducible factor 1α (HIF1α) and E2F1 dependent. Two HIF1α binding sites on miR-17~92 promoter were identified. We also found that long-term hypoxia-induced suppression of miR-17~92 expression could be restored by silencing of p53. Mutation of the p53-binding sites in the miR-17~92 promoter increased miR-17~92 promoter activity in both normoxia and hypoxia. Our findings suggest that the biphasic transcriptional regulation of miR-17~92 during hypoxia is controlled by HIF1/E2F1 and p53 in PASMC: during short-term hypoxia exposure, stabilization of HIF1 and induction of E2F1 induce the transcription of miR-17~92, whereas during long-term hypoxia exposure, hyperphosphorylation of p53 suppresses the expression of miR-17~92. NEW & NOTEWORTHY We showed that the biphasic transcriptional regulation of miR-17~92 during hypoxia is controlled by two distinct mechanisms: during short-term hypoxia exposure, induction of HIF1 and E2F1 upregulates miR-17~92. Longer hypoxia exposure induces hyperphosphorylation of p53 at ser15, which leads to its binding to miR-17~92 promoter and inhibition of its expression. Our findings provide novel insights into the spatiotemporal regulation of miR-17~92 that may play a role in the development of human lung diseases including pulmonary hypertension (PH). [ABSTRACT FROM AUTHOR]

One exercise session can increase subsequent insulin-stimulated glucose uptake (ISGU) by skeletal muscle from rodents and humans of both sexes. We recently found that concurrent mutation of three key sites to prevent their phosphorylation (Ser588, Thr642, and Ser704) on Akt substrate of 160 kDa (AS160; also known as TBC1D4) reduced the magnitude of the enhancement of postexercise ISGU (PEX-ISGU) by muscle from male, but not female rats. However, we did not test the role of individual phosphorylation sites on PEX-ISGU. Accordingly, our current aim was to test whether AS160 Ser704 phosphorylation (pSer704) is required for elevated PEX-ISGU by muscle. AS160-knockout (AS160-KO) rats (female and male) were studied when either in sedentary or 3 h after acute exercise. Adeno-associated virus (AAV) vectors were used to enable muscle expression of wild-type AS160 (AAV-WT-AS160) or AS160 mutated Ser704 to alanine to prevent phosphorylation (AAV-1P-AS160). Paired epitrochlearis muscles from each rat were injected with AAV-WT-AS160 or AAV-1P-AS160. We discovered that regardless of sex 1) AS160 abundance in AS160-KO rats was similar in paired muscles expressing WT-AS160 versus 1P-AS160; 2) muscles from exercised versus sedentary rats had greater ISGU, and PEX-ISGU was slightly greater for muscles expressing 1P-AS160 versus contralateral muscles expressing WT-AS160; and 3) pAS160Thr642 was lower in muscles expressing 1P-AS160 versus paired muscles expressing WT-AS160. These results indicate that pAS160Ser704 was not essential for elevated PEX-ISGU by skeletal muscle from rats of either sex. Furthermore, elimination of the postexercise increase in pAS160Thr642 did not lessen the postexercise effect on ISGU. NEW & NOTEWORTHY: The current study evaluated the role of Akt substrate of 160 kDa (AS160) phosphorylation on Ser704 in increased insulin-stimulated glucose uptake by skeletal muscle after exercise. Adeno-associated virus vectors were engineered to express either wild-type-AS160 or AS160 mutated so that it could not be phosphorylated on Ser704 in paired muscles from AS160-knockout rats. The results demonstrated that AS160 phosphorylation on Ser704 was not essential for exercise-induced elevation in insulin-stimulated glucose uptake by rats of either sex. [ABSTRACT FROM AUTHOR]

Diastolic dysfunction and delayed ventricular repolarization are typically observed in the elderly, but whether these defects are intimately associated with the progressive manifestation of the aging myopathy remains to be determined. In this regard, aging in experimental animals is coupled with increased late Na+ current (INa,L) in cardiomyocytes, raising the possibility that INa,L conditions the modality of electrical recovery and myocardial relaxation of the aged heart. For this purpose, aging male and female wild-type (WT) C57Bl/6 mice were studied together with genetically engineered mice with phosphomimetic (gain of function, GoF) or ablated (loss of function, LoF) mutations of the sodium channel Nav1.5 at Ser571 associated with, respectively, increased and stabilized INa,L. At ∼18 mo of age, WT mice developed prolonged duration of the QT interval of the electrocardiogram and impaired diastolic left ventricular (LV) filling, defects that were reversed by INa,L inhibition. Prolonged repolarization and impaired LV filling occurred prematurely in adult (∼5 mo) GoF mutant mice, whereas these alterations were largely attenuated in aging LoF mutant animals. Ca2+ transient decay and kinetics of myocyte shortening/relengthening were delayed in aged (∼24 mo) WT myocytes, with respect to adult cells. In contrast, delayed Ca2+ transients and contractile dynamics occurred at adult stage in GoF myocytes and further deteriorated in old age. Conversely, myocyte mechanics were minimally affected in aging LoF cells. Collectively, these results document that Nav1.5 phosphorylation at Ser571 and the late Na+ current modulate the modality of myocyte relaxation, constituting the mechanism linking delayed ventricular repolarization and diastolic dysfunction. NEW & NOTEWORTHY: We have investigated the impact of the late Na current (INa,L) on cardiac and myocyte function with aging by using genetically engineered animals with enhanced or stabilized INa,L, due to phosphomimetic or phosphoablated mutations of Nav1.5. Our findings support the notion that phosphorylation of Nav1.5 at Ser571 prolongs myocardial repolarization and impairs diastolic function, contributing to the manifestations of the aging myopathy. [ABSTRACT FROM AUTHOR]

Prior studies showed that epidermal growth factor (EGF) inhibits vasopressin-stimulated osmotic water permeability in the renal collecting duct. Here, we investigated the underlying mechanism. Using isolated perfused rat inner medullary collecting ducts (IMCDs), we found that the addition of EGF to the peritubular bath significantly decreased 1-deamino-8-D-arginine vasopressin (dDAVP)-stimulated water permeability, confirming prior observations. The inhibitory effect of EGF on water permeability was associated with a reduction in intracellular cAMP levels and protein kinase A (PKA) activity. Using phospho-specific antibodies and immunoblotting in IMCD suspensions, we showed that EGF significantly reduces phosphorylation of AQP2 at Ser264 and Ser269. This effect was absent when 8-cpt-cAMP was used to induce AQP2 phosphorylation, suggesting that EGF's inhibitory effect was at a pre-cAMP step. Immunofluorescence labeling of microdissected IMCDs showed that EGF significantly reduced apical AQP2 abundance in the presence of dDAVP. To address what protein kinase might be responsible for Ser269 phosphorylation, we used Bayesian analysis to integrate multiple-omic datasets. Thirteen top-ranked protein kinases were subsequently tested by in vitro phosphorylation experiments for their ability to phosphorylate AQP2 peptides using a mass spectrometry readout. The results show that the PKA catalytic-a subunit increased phosphorylation at Ser256, Ser264, and Ser269. None of the other kinases tested phosphorylated Ser269. In addition, H-89 and PKI strongly inhibited dDAVP-stimulated AQP2 phosphorylation at Ser269. These results indicate that EGF decreases the water permeability of the IMCD by inhibiting cAMP production, thereby inhibiting PKA and decreasing AQP2 phosphorylation at Ser269, a site previously shown to regulate AQP2 endocytosis. [ABSTRACT FROM AUTHOR]

Prior studies showed that epidermal growth factor (EGF) inhibits vasopressin-stimulated osmotic water permeability in the renal collecting duct. Here, we investigated the underlying mechanism. Using isolated perfused rat inner medullary collecting ducts (IMCDs), we found that the addition of EGF to the peritubular bath significantly decreased 1-deamino-8-D-arginine vasopressin (dDAVP)-stimulated water permeability, confirming prior observations. The inhibitory effect of EGF on water permeability was associated with a reduction in intracellular cAMP levels and protein kinase A (PKA) activity. Using phospho-specific antibodies and immunoblotting in IMCD suspensions, we showed that EGF significantly reduces phosphorylation of AQP2 at Ser264 and Ser269. This effect was absent when 8-cpt-cAMP was used to induce AQP2 phosphorylation, suggesting that EGF's inhibitory effect was at a pre-cAMP step. Immunofluorescence labeling of microdissected IMCDs showed that EGF significantly reduced apical AQP2 abundance in the presence of dDAVP. To address what protein kinase might be responsible for Ser269 phosphorylation, we used Bayesian analysis to integrate multiple-omic datasets. Thirteen top-ranked protein kinases were subsequently tested by in vitro phosphorylation experiments for their ability to phosphorylate AQP2 peptides using a mass spectrometry readout. The results show that the PKA catalytic-a subunit increased phosphorylation at Ser256, Ser264, and Ser269. None of the other kinases tested phosphorylated Ser269. In addition, H-89 and PKI strongly inhibited dDAVP-stimulated AQP2 phosphorylation at Ser269. These results indicate that EGF decreases the water permeability of the IMCD by inhibiting cAMP production, thereby inhibiting PKA and decreasing AQP2 phosphorylation at Ser269, a site previously shown to regulate AQP2 endocytosis. [ABSTRACT FROM AUTHOR]

Vasopressin regulates water homeostasis via the V2 receptor in the kidney at least in part through protein kinase A (PKA) activation. Vasopressin, through an unknown pathway, upregulates the activity and phosphorylation of Na+-Cl- cotransporter (NCC) and Na+-K+-2Cl- cotransporter 2 (NKCC2) by Ste20-related proline/alanine-rich kinase (SPAK) and oxidative stress-responsive kinase 1 (OSR1), which are regulated by the with-no-lysine kinase (WNK) family. Phosphorylation of WNK4 at PKA consensus motifs may be involved. Inhibitor 1 (I1), a protein phosphatase 1 (PP1) inhibitor, may also play a role. In human embryonic kidney (HEK)-293 cells, we assessed the phosphorylation of WNK4, SPAK, NCC, or NKCC2 in response to forskolin or desmopressin. WNK4 and cotransporter phosphorylation were studied in desmopressin-infused WNK4-/- mice and in tubule suspensions. In HEK-293 cells, only wild-type WNK4 but not WNK1, WNK3, or a WNK4 mutant lacking PKA phosphorylation motifs could upregulate SPAK or cotransporter phosphorylation in response to forskolin or desmopressin. I1 transfection maximized SPAK phosphorylation in response to forskolin in the presence of WNK4 but not of mutant WNK4 lacking PP1 regulation. We observed direct PP1 regulation of NKCC2 dephosphorylation but not of NCC or SPAK in the absence of WNK4. WNK4-/- mice with desmopressin treatment did not increase SPAK/OSR1, NCC, or NKCC2 phosphorylation. In stimulated tubule suspensions from WNK4-/- mice, upregulation of pNKCC2 was reduced, whereas upregulation of SPAK phosphorylation was absent. These findings suggest that WNK4 is a central node in which kinase and phosphatase signaling converge to connect cAMP signaling to the SPAK/OSR1-NCC/NKCC2 pathway. NEW & NOTEWORTHY With-no-lysine kinases regulate the phosphorylation and activity of the Na+-Cl- and Na+-K+-2Cl- cotransporters. This pathway is modulated by arginine vasopressin (AVP). However, the link between AVP and WNK signaling remains unknown. Here, we show that AVP activates WNK4 through increased phosphorylation at putative protein kinase A-regulated sites and decreases its dephosphorylation by protein phosphatase 1. This work increases our understanding of the signaling pathways mediating AVP actions in the kidney. [ABSTRACT FROM AUTHOR]

The with-no-lysine kinase 4 (WNK4)-sterile 20/SPS-1-related proline/alanine-rich kinase (SPAK)/oxidative stress-responsive kinase 1 (OSR1) pathway mediates activating phosphorylation of the furosemide-sensitive Na+-K+-2Cl- cotransporter (NKCC2) and the thiazide-sensitive NaCl cotransporter (NCC). The commonly used pT96/pT101-pNKCC2 antibody cross-reacts with pT53-NCC in mice on the C57BL/6 background due to a five amino acid deletion. We generated a new C57BL/6-specific pNKCC2 antibody (anti-pT96-NKCC2) and tested the hypothesis that the WNK4-SPAK/OSR1 pathway strongly regulates the phosphorylation of NCC but not NKCC2. In C57BL/6 mice, anti-pT96-NKCC2 detected pNKCC2 and did not cross-react with NCC. Abundances of pT96-NKCC2 and pT53-NCC were evaluated in Wnk4-/-, Osr1-/-, Spak-/-, and Osr1-/-/Spak-/- mice and in several models of the disease familial hyperkalemic hypertension (FHHt) in which the CUL3-KLHL3 ubiquitin ligase complex that promotes WNK4 degradation is dysregulated (Cul3+/-/Δ9-Klhl3-/-, and Klhl3R528H/R528H). All mice were on the C57BL/6 background. In Wnk4-/- mice, pT53-NCC was almost absent but pT96-NKCC2 was only slightly lower. pT53-NCC was almost absent in Spak-/- and Osr1-/-/Spak-/- mice, but pT96-NKCC2 abundance did not differ from controls. pT96-NKCC2/total NKCC2 was slightly lower in Osr1-/- and Osr1-/-/Spak-/- mice. WNK4 expression colocalized not only with NCC but also with NKCC2 in Klhl3-/- mice, but pT96-NKCC2 abundance was unchanged. Consistent with this, furosemide-induced urinary Na+ excretion following thiazide treatment was similar between Klhl3-/- and controls. pT96-NKCC2 abundance was also unchanged in the other FHHt mouse models. Our data show that disruption of the WNK4-SPAK/OSR1 pathway only mildly affects NKCC2 phosphorylation, suggesting a role for other kinases in NKCC2 activation. In FHHt models NKCC2 phosphorylation is unchanged despite higher WNK4 abundance, explaining the thiazide sensitivity of FHHt. [ABSTRACT FROM AUTHOR]

Tolvaptan, a vasopressin antagonist selective for the V2-subtype vasopressin receptor (V2R), is widely used in the treatment of hyponatremia and autosomal-dominant polycystic kidney disease (ADPKD). Its effects on signaling in collecting duct cells have not been fully characterized. Here, we perform RNA-seq in a collecting duct cell line (mpkCCD). The data show that tolvaptan inhibits the expression of mRNAs that were previously shown to be increased in response to vasopressin including aquaporin-2, but also reveals mRNA changes that were not readily predictable and suggest off-target actions of tolvaptan. One such action is activation of the MAPK kinase (ERK1/ERK2) pathway. Prior studies have shown that ERK1/ERK2 activation is essential in the regulation of a variety of cellular and physiological processes and can be associated with cell proliferation. In immunoblotting experiments, we demonstrated that ERK1/ERK2 phosphorylation in mpkCCD cells was significantly reduced by vasopressin, in contrast to the increases seen in non-collecting-duct cells overexpressing V2R in prior studies. We also found that tolvaptan has a strong effect to increase ERK1/ERK2 phosphorylation in the presence of vasopressin and that tolvaptan's effect to increase ERK1/ERK2 phosphorylation is absent in mpkCCD cells in which both protein kinase A (PKA)-catalytic subunits have been deleted. Thus, it appears that the tolvaptan effect to increase ERK activation is PKA-dependent and is not due to an off-target effect of tolvaptan. We conclude that in cells expressing V2R at endogenous levels: 1) vasopressin decreases ERK1/ERK2 activation; 2) in the presence of vasopressin, tolvaptan increases ERK1/ERK2 activation; and 3) these effects are PKA-dependent. [ABSTRACT FROM AUTHOR]

Electroneutral NaCl transport by Na+/H+ exchanger 3 (NHE3, SLC9A3) is the major Na+ absorptive mechanism in the intestine and decreased NHE3 activity contributes to diarrhea. Patients with diabetes often experience gastrointestinal adverse effects and medications are often a culprit for chronic diarrhea in type 2 diabetes (T2D). We have shown previously that metformin, the most widely prescribed drug for the treatment of T2D, induces diarrhea by inhibition of Naþ/Hþ exchanger 3 (NHE3) in rodent models of T2D. Metformin was shown to activate AMP-activated protein kinase (AMPK), but AMPK-independent glycemic effects of metformin are also known. The current study is undertaken to determine whether metformin inhibits NHE3 by activation of AMPK and the mechanism by which NHE3 is inhibited by AMPK. Inhibition of NHE3 by metformin was abolished by knockdown of AMPK-α1 or AMPK-α2. AMPK activation by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) phosphorylated NHE3 at S555. S555 is the primary site of phosphorylation by protein kinase A (PKA), but AMPK phosphorylated S555 independently of PKA. Using Mass spectrometry, we found S563 as a newly recognized phosphorylation site in NHE3. Altering either S555 or S563 to Ala was sufficient to block the inhibition of NHE3 activity by AMPK. NHE3 inhibition is dependent on ubiquitination by the E3 ubiquitin ligase Nedd4-2 and metformin was shown to induce NHE3 internalization via Nedd4-2-mediated ubiquitination. AICAR did not increase NHE3 ubiquitination when S555 or S563 was mutated. We conclude that AMPK activation inhibits NHE3 activity and NHE3 inhibition is associated with phosphorylation of NHE3 at S555 and S563. NEW & NOTEWORTHY: We show that AMP-activated protein kinase (AMPK) phosphorylates NHE3 at S555 and S563 to inhibit NHE3 activity in intestinal epithelial cells. Phosphorylation of NHE3 by AMPK is necessary for ubiquitination of NHE3. [ABSTRACT FROM AUTHOR]

The solute carrier family 12A (SLC12A) superfamily of membrane transporters modulates the movement of cations coupled with chloride across the membrane. In doing so, these cotransporters are involved in numerous aspects of human physiology: cell volume regulation, ion homeostasis, blood pressure regulation, and neurological action potential via intracellular chloride concentration modulation. Their physiological characterization has been largely studied; however, understanding the mechanics of their function and the relevance of structural domains or specific amino acids has been a pending task. In recent years, singleparticle cryogenic electron microscopy (cryo-EM) has been successfully applied to members of the SLC12A family including all K+:Cl- cotransporters (KCCs), Na+:K+:2Cl- cotransporter NKCC1, and recently Na+:Cl- cotransporter (NCC); revealing structural elements that play key roles in their function. The present review analyzes the data provided by these cryo-EM reports focusing on structural domains and specific amino acids involved in ion binding, domain interactions, and other important SCL12A structural elements. A comparison of cryo-EM data from NKCC1 and KCCs is presented in the light of the two recent NCC cryo-EM studies, to propose insight into structural elements that might also be found in NCC and are necessary for its proper function. In the final sections, the importance of key coordination residues for substrate specificity and their implication on various pathophysiological conditions and genetic disorders is reviewed, as this could provide the basis to correlate structural elements with the development of novel and selective treatments, as well as mechanistic insight into the function and regulation of cationcoupled chloride cotransporters (CCCs). [ABSTRACT FROM AUTHOR]

Vasopressin controls renal water excretion through actions to regulate aquaporin-2 (AQP2) trafficking, transcription, and degradation. These actions are in part dependent on vasopressin-induced phosphorylation changes in collecting duct cells. Although most efforts have focused on the phosphorylation of AQP2 itself, phosphoproteomic studies have identified many vasopressinregulated phosphorylation sites in proteins other than AQP2. The goal of this bioinformatics-based review is to create a compendium of vasopressin-regulated phosphorylation sites with a focus on those that are seen in both native rat inner medullary collecting ducts and cultured collecting duct cells from the mouse (mpkCCD), arguing that these sites are the best candidates for roles in AQP2 regulation. This analysis identified 51 vasopressin-regulated phosphorylation sites in 45 proteins. We provide resource web pages at https://esbl.nhlbi.nih.gov/Databases/AVP-Phos/and https://esbl.nhlbi.nih.gov/AVP-Network/, listing the phosphorylation sites and describing annotated functions of each of the vasopressin-targeted phosphoproteins. Among these sites are 23 consensus protein kinase A (PKA) sites that are increased in response to vasopressin, consistent with a central role for PKA in vasopressin signaling. The remaining sites are predicted to be phosphorylated by other kinases, most notably ERK1/2, which accounts for decreased phosphorylation at sites with a X-p(S/T)-P-X motif. Additional protein kinases that undergo vasopressininduced changes in phosphorylation are Camkk2, Cdk18, Erbb3, Mink1, and Src, which also may be activated directly or indirectly by PKA. The regulated phosphoproteins are mapped to processes that hypothetically can account for vasopressin-mediated control of AQP2 trafficking, cytoskeletal alterations, and Aqp2 gene expression, providing grist for future studies. [ABSTRACT FROM AUTHOR]

Sympathetic regulation of the Kv4.2 transient outward potassium current (Ito) is critical for the acute electrical and contractile response of the myocardium under physiological and pathological conditions. Previous studies have suggested that KChIP2, the key auxiliary subunit of Kv4 channels, is required for the sympathetic regulation of Kv4.2 current densities. Of interest, Kv4.2 and KChIP2, and key components mediating acute sympathetic signaling transduction are present in lipid rafts, which are profoundly involved in regulation of Ito densities in rat ventricular myocytes. However, little is known about the mechanisms of Kv4.2-raft association and its connection with acute sympathetic regulation. With the aid of high-resolution fluorescent microscope, we demonstrated that KChIP2 assisted Kv4.2 localization in lipid rafts in HEK293 cells. Moreover, PKA-mediated Kv4.2 phosphorylation, the downstream signaling event of acute sympathetic stimulation, induced dissociation between Kv4.2 and KChIP2, resulting in Kv4.2 shifting out of lipid rafts in KChIP2-expressed HEK293. The mutation that mimics Kv4.2 phosphorylation by PKA (K4.2-S552D) similarly disrupted Kv4.2 interaction with KChIP2 and also decreased the surface stability of Kv4.2. The attenuated Kv4.2-KChIP2 interaction was also observed in native neonatal rat ventricular myocytes (NRVMs) upon acute adrenergic stimulation with phenylephrine (PE). Furthermore, PE stimulation decreased Kv4.2 location at lipid rafts and induced internalization of Kv4.2 as well as the effect of lipid rafts disruption. In conclusion, KChIP2 contributes to targeting Kv4.2 to lipid rafts. Acute adrenergic stimulation induces Kv4.2-KChIP2 dissociation, leading to Kv4.2 out of lipid rafts and internalization, reinforcing the critical role of Kv4.2-lipid raft association in the essential physiological response of Ito to acute sympathetic regulation. [ABSTRACT FROM AUTHOR]

The biology of the cyclin-dependent kinase-like (CDKL) kinase family remains enigmatic. Contrary to their nomenclature, CDKLs do not rely on cyclins for activation and are not involved in cell cycle regulation. Instead, they share structural similarities with mitogen-activated protein kinases and glycogen synthase kinase-3, although their specific functions and associated signaling pathways are still unknown. Previous studies have shown that the activation of CDKL5 kinase contributes to the development of acute kidney injury (AKI) by suppressing the protective SOX9-dependent transcriptional program in tubular epithelial cells. In the current study, we measured the functional activity of all five CDKL kinases and discovered that, in addition to CDKL5, CDKL1 is also activated in tubular epithelial cells during AKI. To explore the role of CDKL1, we generated a germline knockout mouse that exhibited no abnormalities under normal conditions. Notably, when these mice were challenged with bilateral ischemia-reperfusion and rhabdomyolysis, they were found to be protected from AKI. Further mechanistic investigations revealed that CDKL1 phosphorylates and destabilizes SOX11, contributing to tubular dysfunction. In summary, this study has unveiled a previously unknown CDKL1-SOX11 axis that drives tubular dysfunction during AKI. NEW & NOTEWORTHY Identifying and targeting pathogenic protein kinases holds potential for drug discovery in treating acute kidney injury. Our study, using novel germline knockout mice, revealed that Cdkl1 kinase deficiency does not affect mouse viability but provides protection against acute kidney injury. This underscores the importance of Cdkl1 kinase in kidney injury and supports the development of targeted small-molecule inhibitors as potential therapeutics.

Dietary potassium deficiency causes stimulation of sodium reabsorption leading to an increased risk in blood pressure elevation. The distal convoluted tubule (DCT) is the main rheostat linking plasma K + levels to the activity of the Na-Cl cotransporter (NCC). This occurs through basolateral membrane potential sensing by inwardly rectifying K + channels (Kir4.1/5.1); decrease in intracellular Cl - ; activation of WNK4 and interaction and phosphorylation of STE20/SPS1-related proline/alanine-rich kinase (SPAK); binding of calcium-binding protein 39 (cab39) adaptor protein to SPAK, leading to its trafficking to the apical membrane; and SPAK binding, phosphorylation, and activation of NCC. As kidney-specific with-no-lysine kinase 1 (WNK1) isoform (KS-WNK1) is another participant in this pathway, we examined its function in NCC regulation. We eliminated KS-WNK1 specifically in the DCT and demonstrated increased expression of WNK4 and long WNK1 (L-WNK1) and increased phosphorylation of NCC. As in other KS-WNK1 models, the mice were not hyperkalemic. Although wild-type mice under low-dietary K + conditions demonstrated increased NCC phosphorylation, the phosphorylation levels of the transporter, already high in KS-WNK1, did not change under the low-K + diet. Thus, in the absence of KS-WNK1, the transporter lost its sensitivity to low plasma K + . We also show that under low K + conditions, in the absence of KS-WNK1, there was no formation of WNK bodies. These bodies were observed in adjacent segments, not affected by the targeting of KS-WNK1. As our data are overall consistent with those of the global KS-WNK1 knockout, they indicate that the DCT is the predominant segment affecting the salt transport regulated by KS-WNK1. NEW & NOTEWORTHY In this paper, we show that KS-WNK1 is a critical component of the distal convoluted tubule (DCT) K + switch pathway. Its deletion results in an inability of the DCT to sense changes in plasma potassium. Absence of KS-WNK1 leads to abnormally high levels of WNK4 and L-WNK1 in the DCT, resulting in increased Na-Cl phosphorylation and function. Our data are consistent with KS-WNK1 targeting WNK4 and L-WNK1 to degradation.

We have previously observed that prolonged administration of rapamycin, an inhibitor targeting the mammalian target of rapamycin complex (mTORC)1, partially reduced hypertension and alleviated kidney inflammation in Dahl salt-sensitive (SS) rats. In contrast, treatment with PP242, an inhibitor affecting both mTORC1/mTORC2, not only completely prevented hypertension but also provided substantial protection against kidney injury. Notably, PP242 exhibited potent natriuretic effects that were not evident with rapamycin. The primary objective of this study was to pinpoint the specific tubular sites responsible for the natriuretic effect of PP242 in SS rats subjected to either 0.4% NaCl (normal salt) or 4.0% NaCl (high salt) diet. Acute effects of PP242 on natriuretic, diuretic, and kaliuretic responses were determined in unanesthetized SS rats utilizing benzamil, furosemide, or hydrochlorothiazide [inhibitors of epithelial Na + channel (ENaC), Na-K-2Cl cotransporter (NKCC2), or Na-Cl cotransporter (NCC), respectively] either administered alone or in combination. The findings indicate that the natriuretic effects of PP242 in SS rats stem predominantly from the inhibition of NCC and a reduction of ENaC open probability. Molecular analysis revealed that mTORC2 regulates NCC activity through protein phosphorylation and ENaC activity through proteolytic cleavage in vivo. Evidence also indicated that PP242 also prevents the loss of K + associated with the inhibition of NCC. These findings suggest that PP242 may represent an improved therapeutic approach for antihypertensive intervention, potentially controlling blood pressure and mitigating kidney injury in salt-sensitive human subjects. NEW & NOTEWORTHY This study explored mechanisms underlying the natriuretic effects of mammalian target of rapamycin protein complex 2 inhibition using PP242 and revealed both epithelial Na + channel and Na-Cl cotransporter in the distal tubular segments were potentially inhibited. These observations, with prior lab evidence, indicate that PP242 prevents hypertension via its potent inhibitory effects on these specific sodium transporters and by reducing renal immune responses. This dual action, coupled with potassium sparing effects, suggests an improved approach for managing hypertension and associated kidney damage.

RhoA and its effectors, the transcriptional coactivators myocardin-related transcription factor (MRTF) and serum response factor (SRF), control epithelial phenotype and are indispensable for profibrotic epithelial reprogramming during fibrogenesis. Context-dependent control of RhoA and fibrosis-associated changes in its regulators, however, remain incompletely characterized. We previously identified the guanine nucleotide exchange factor GEF-H1 as a central mediator of RhoA activation in renal tubular cells exposed to inflammatory or fibrotic stimuli. Here we found that GEF-H1 expression and phosphorylation were strongly elevated in two animal models of fibrosis. In the Unilateral Ureteral Obstruction mouse kidney fibrosis model, GEF-H1 was upregulated predominantly in the tubular compartment. GEF-H1 was also elevated and phosphorylated in a rat pulmonary artery banding (PAB) model of right ventricular fibrosis. Prolonged stimulation of LLC-PK 1 tubular cells with tumor necrosis factor (TNF)-α or transforming growth factor (TGF)-β1 increased GEF-H1 expression and activated a luciferase-coupled GEF-H1 promoter. Knockdown and overexpression studies revealed that these effects were mediated by RhoA, cytoskeleton remodeling, and MRTF, indicative of a positive feedback cycle. Indeed, silencing endogenous GEF-H1 attenuated activation of the GEF-H1 promoter. Of importance, inhibition of MRTF using CCG-1423 prevented GEF-H1 upregulation in both animal models. MRTF-dependent increase in GEF-H1 was prevented by inhibition of the transcription factor Sp1, and mutating putative Sp1 binding sites in the GEF-H1 promoter eliminated its MRTF-dependent activation. As the GEF-H1/RhoA axis is key for fibrogenesis, this novel MRTF/Sp1-dependent regulation of GEF-H1 abundance represents a potential target for reducing renal and cardiac fibrosis. NEW & NOTEWORTHY We show that expression of the RhoA regulator GEF-H1 is upregulated in tubular cells exposed to fibrogenic cytokines and in animal models of kidney and heart fibrosis. We identify a pathway wherein GEF-H1/RhoA-dependent MRTF activation through its noncanonical partner Sp1 upregulates GEF-H1. Our data reveal the existence of a positive feedback cycle that enhances Rho signaling through control of both GEF-H1 activation and expression. This feedback loop may play an important role in organ fibrosis.

Hypertension affects approximately one in two United States adults and sex plays an important role in the pathogenesis of hypertension. The Na + -Cl - cotransporter (NCC), regulated by a kinase network including with-no-lysine kinase (WNK)1 and WNK4, STE20/SPS1-related proline alanine-rich kinase (SPAK), and oxidative stress response 1 (OxSR1), is critical to Na + reabsorption and blood pressure regulation. Dietary salt differentially modulates NCC in salt-sensitive and salt-resistant rats, in part by modulation of WNK/SPAK/OxSR1 signaling. In this study, we tested the hypothesis that sex-dependent differences in NCC regulation contribute to the development of the salt sensitivity of blood pressure using male and female Sprague-Dawley (SD), Dahl salt-resistant (DSR), and Dahl salt-sensitive (DSS) rats. In normotensive salt-resistant SD and DSR rats, a high-salt diet evoked significant decreases in NCC activity, expression, and phosphorylation. In males, these changes were associated with no change in WNK1 expression, a decrease in WNK4 levels, and suppression of SPAK/OxSR1 expression and phosphorylation. In contrast, in females, there was decreased NCC activity associated with suppression of SPAK/OxSR1 expression and phosphorylation. In hypertensive DSS rats, the ability of females to suppress NCC (in opposition to males) via a SPAK/OxSR1 mechanism likely contributes to their lower magnitude of salt-sensitive hypertension. Collectively, our findings support the existence of sex differences in male versus female rats with NCC regulation during dietary salt intake involving suppression of WNK4 expression in male rats only and the involvement of SPAK/OxSR1 signaling in both males and females. NEW & NOTEWORTHY NCC regulation is sex dependent. In normotensive male and female Sprague-Dawley and Dahl salt-resistant rats, which exhibit dietary Na + -evoked NCC suppression, male rats exhibit decreased WNK4 expression and decreased SPAK and OxSR1 levels, whereas female rats only suppress SPAK and OxSR1. In hypertensive Dahl salt-sensitive rats, the ability of females to suppress NCC (in opposition to males) via a SPAK/OxSR1 mechanism likely contributes to their lower magnitude of salt-sensitive hypertension.

Although the unfolded protein response (UPR) contributes to survival by removing misfolded proteins, endoplasmic reticulum (ER) stress also activates proapoptotic pathways. Changed sensitivity to normal developmental stimuli may underlie observed cardiomyocyte apoptosis in the healthy perinatal heart. We determined in vitro sensitivity to thapsigargin in sheep cardiomyocytes from four perinatal ages. In utero cardiac activation of ER stress and apoptotic pathways was determined at these same ages. Thapsigargin-induced phosphorylation of eukaryotic initiation factor 2 (EIF2A) was decreased by 72% between 135 and 143 dGA ( P = 0.0096) and remained low at 1 dPN ( P = 0.0080). Conversely, thapsigargin-induced caspase cleavage was highest around the time of birth: cleaved caspase 3 was highest at 1 dPN (3.8-fold vs. 135 dGA, P = 0.0380; 7.8-fold vs. 5 dPN, P = 0.0118), cleaved caspase 7 and cleaved caspase 12 both increased between 135 and 143 dGA (25-fold and 6.9-fold respectively, both P < 0.0001) and remained elevated at 1 dPN. Induced apoptosis, measured by TdT-mediated dUTP nick-end labeling (TUNEL) assay, was highest around the time of birth ( P < 0.0001). There were changes in myocardial ER stress pathway components in utero. Glucose (78 kDa)-regulated protein (GRP78) protein levels were high in the fetus and declined after birth ( P < 0.0001). EIF2A phosphorylation was profoundly depressed at 1 dPN (vs. 143 dGA, P = 0.0113). In conclusion, there is dynamic regulation of ER proteostasis, ER stress, and apoptosis cascade in the perinatal heart. Apoptotic signaling is more readily activated in fetal cardiomyocytes near birth, leading to widespread caspase cleavage in the newborn heart. These pathways are important for the regulation of normal maturation in the healthy perinatal heart. NEW & NOTEWORTHY Cardiomyocyte apoptosis occurs even in the healthy, normally developing perinatal myocardium. As cardiomyocyte number is a critical contributor to heart health, the sensitivity of cardiomyocytes to endoplasmic reticulum stress leading to apoptosis is an important consideration. This study suggests that the heart has less robust protective mechanisms in response to endoplasmic reticulum stress immediately before and after birth, and that more cardiomyocyte death can be induced by stress in this period.

The cardiovascular benefits of regular exercise are unequivocal, yet patients with type 2 diabetes respond poorly to exercise due to a reduced cardiac reserve. The contractile response of diabetic cardiomyocytes to β-adrenergic stimulation is attenuated, which may result in altered myofilament calcium sensitivity and posttranslational modifications of cardiac troponin I (cTnI). Treadmill running increases myofilament calcium sensitivity in nondiabetic rats, and thus we hypothesized that endurance training would increase calcium sensitivity of diabetic cardiomyocytes and alter site-specific phosphorylation of cTnI. Calcium sensitivity, or pCa50, was measured in Zucker diabetic fatty (ZDF), nondiabetic (nDM), and diabetic (DM) rat hearts after 8 wk of either a sedentary (SED) or progressive treadmill running (TR) intervention. Skinned cardiomyocytes were connected to a capacitance-gauge transducer and a torque motor to measure force as a function of pCa (-log[Ca2+]). Specific phospho-sites on cTnI and O-GlcNAcylation were quantified by immunoblot and total protein phosphorylation by fluorescent gel staining (ProQ Diamond). The novel finding in this study was that training increased pCa50 in both DM and nDM cardiomyocytes (P = 0.009). Phosphorylation of cTnI amino acid residues Ser23/24, a crucial protein kinase A site, and Threonine (Thr)144 was lower in DM hearts, but there was no effect of training on site-specific phosphorylation. In addition, total phosphorylation and O-GlcNAcylation levels were not different between SED and TR groups. These findings suggest that regular exercise may benefit the diabetic heart by specifically targeting myofilament contractile function. NEW & NOTEWORTHY We examined the effects of training on the myofilament calcium in diabetic rat hearts. After 8 wk of treadmill running, both nondiabetic and diabetic cardiomyocytes had increased myofilament calcium sensitivity compared with their sedentary counterparts, but there was no effect of training on the phosphorylation or O-GlcNAcylation status of myofilament proteins measured in this study. These data highlight one potential mechanism capable of reversing, in part, reduced cardiac reserve in the diabetic heart. [ABSTRACT FROM AUTHOR]

In mice, exercise is suggested to activate the mechanistic target of rapamycin complex 2 (mTORC2) in skeletal muscle, and mTORC2 is required for normal muscle glucose uptake during exercise. Whether this translates to human skeletal muscle and what signaling pathways facilitate the exercise-induced mTORC2 activation is unknown. We herein tested the hypothesis that exercise increases mTORC2 activity in human skeletal muscle and investigated if β2-adrenergic receptor (AR) activation mediates exercise-induced mTORC2 activation. We examined several mTORC2 activity readouts (p-NDRG1 Thr346, p-Akt Ser473, p-mTOR S2481, and p-Akt Thr450) in human skeletal muscle biopsies after uphill walking or cycling exercise. In mouse muscles, we assessed mTORC2 activity readouts following acute activation of muscle b2-adrenergic or GS signaling and during in vivo and ex vivo muscle contractions. Exercise increased phosphorylation of NDRG1 Thr346 in human soleus, gastrocnemius, and vastus lateralis muscle, without changing p-Akt Ser473, p-Akt Thr450, and p-mTOR Ser2481. In mouse muscle, stimulation of β2-adrenergic or GS signaling and ex vivo contractions failed to increase p-NDRG1 Thr346, whereas in vivo contractions were sufficient to induce p-NDRG1 Thr346. In conclusion, the mTORC2 activity readout p-NDRG1 Thr346 is a novel exercise-responsive signaling protein in human skeletal muscle. Notably, contraction-induced p-NDRG1 Thr346 appears to require a systemic factor. Unlike exercise, and in contrast to published data obtained in cultured muscles cells, stimulation of b2-adrenergic signaling is not sufficient to trigger NDRG1 phosphorylation in mature mouse skeletal muscle. [ABSTRACT FROM AUTHOR]

Following anabolic stimuli (mechanical loading and/or amino acid provision), the mechanistic target of rapamycin complex 1 (mTORC1), a master regulator of protein synthesis, translocates toward the cell periphery. However, it is unknown if mTORC1- mediated phosphorylation events occur in these peripheral regions or before translocation (i.e., in central regions). We therefore aimed to determine the cellular location of a mTORC1-mediated phosphorylation event, RPS6Ser240/244, in human skeletal muscle following anabolic stimuli. Fourteen young, healthy males either ingested a protein-carbohydrate beverage (0.25 g/kg protein and 0.75 g/kg carbohydrate) alone [n = 7; 23 ± 5 yr; 76.8 ± 3.6 kg; and 13.6 ± 3.8% body fat (BF), FED] or following a whole body resistance exercise bout (n = 7; 22 ± 2 yr; 78.1 ± 3.6 kg; and 12.2 ± 4.9%BF, EXFED). Vastus lateralis muscle biopsies were obtained at rest (PRE) and 120 and 300 min following anabolic stimuli. RPS6Ser240/244 phosphorylation measured by immunofluorescent staining or immunoblot was positively correlated (r = 0.76, P < 0.001). Peripheral staining intensity of p-RPS6Ser240/244 increased above PRE in both FED and EXFED at 120 min (~54% and ~138%, respectively, P < 0.05) but was greater in EXFED at both poststimuli time points (P < 0.05). The peripheral-to-central ratio of p-RPS6240/244 staining displayed a similar pattern, even when corrected for total RPS6 distribution, suggesting RPS6 phosphorylation occurs to a greater extent in the periphery of fibers. Moreover, p-RPS6Ser240/244 intensity within paxillin-positive regions, a marker of focal adhesion complexes, was elevated at 120 min irrespective of stimulus (P = 0.006) before returning to PRE at 300 min. These data confirm that RPS6Ser240/244 phosphorylation occurs in the region of human muscle fibers to which mTOR translocates following anabolic stimuli and identifies focal adhesion complexes as a potential site of mTORC1 regulation in vivo. [ABSTRACT FROM AUTHOR]

Pulmonary microvascular endothelial cells (PMVECs) uniquely express an α1G-subtype of voltage-gated T-type Ca2+ channel. We have previously revealed that the α1G channel functions as a background Ca2+ entry pathway that is critical for the cell proliferation, migration, and angiogenic potential of PMVECs, a novel function attributed to the coupling between α1G-mediated Ca2+ entry and constitutive Akt phosphorylation and activation. Despite this significance, mechanism(s) that link the α1G-mediated Ca2+ entry to Akt phosphorylation remain incompletely understood. In this study, we demonstrate that Ca2+/calmodulin-dependent protein kinase (CaMK) 4 serves as a downstream effector of the α1G-mediated Ca2+ entry to promote the angiogenic potential of PMVECs. Notably, CaMK2 and CaMK4 are both expressed in PMVECs. Pharmacological blockade or genetic knockdown of the α1G channel led to a significant reduction in the phosphorylation level of CaMK4 but not the phosphorylation level of CaMK2. Pharmacological inhibition as well as genetic knockdown of CaMK4 significantly decreased cell proliferation, migration, and network formation capacity in PMVECs. However, CaMK4 inhibition or knockdown did not alter Akt phosphorylation status in PMVECs, indicating that α1G/Ca2+/CaMK4 is independent of the α1G/Ca2+/Akt pathway in sustaining the cells' angiogenic potential. Altogether, these findings suggest a novel α1G-CaMK4 signaling complex that regulates the Ca2+-dominated angiogenic potential in PMVECs. [ABSTRACT FROM AUTHOR]

G protein-coupled receptors (GPCRs) are ubiquitously expressed cell surface receptors that mediate numerous physiological responses and are highly druggable. Upon activation, GPCRs rapidly couple to heterotrimeric G proteins and are then phosphorylated and internalized from the cell surface. Recent studies indicate that GPCRs not only localize at the plasma membrane but also exist in intracellular compartments where they are competent to signal. Intracellular signaling by GPCRs is best described to occur at endosomes. Several studies have elegantly documented endosomal GPCR-G protein and GPCR-β-arrestin signaling. Besides phosphorylation, GPCRs are also posttranslationally modified with ubiquitin. GPCR ubiquitination has been studied mainly in the context of receptor endosomal-lysosomal trafficking. However, new studies indicate that ubiquitination of endogenous GPCRs expressed in endothelial cells initiates the assembly of an intracellular p38 mitogen-activated kinase signaling complex that promotes inflammatory responses from endosomes. In this mini-review, we discuss emerging discoveries that provide critical insights into the function of ubiquitination in regulating GPCR inflammatory signaling at endosomes.

Feeding and resistance exercise stimulate myofibrillar protein synthesis (MPS) rates in healthy adults. This anabolic characterization of "healthy adults" has been namely focused on males. Therefore, the purpose of this study was to examine the temporal responses of MPS and anabolic signaling to resistance exercise alone or combined with the ingestion of protein in postmenopausal females and compare postabsorptive rates with young females. Sixteen females [60 ± 7 yr; body mass index (BMI) = 26 ± 12 kg·m -2 ] completed an acute bout of unilateral resistance exercise before consuming either: a fortified whey protein supplement (WHEY) or water. Participants received primed continuous infusions of L-[ ring - 13 C 6 ]phenylalanine with bilateral muscle biopsies before and after treatment ingestion at 2 h and 4 h in nonexercised and exercised legs. Resistance exercise transiently increased MPS above baseline at 0-2 h in the water condition ( P = 0.007). Feeding after resistance exercise resulted in a late phase (2-4 h) increase in MPS in the WHEY condition ( P = 0.005). In both conditions, resistance exercise did not enhance the cumulative (0-4 h) MPS response. In the nonexercised leg, MPS did not differ at 0-2 h, 2-4 h, or 0-4 h of the measurement periods (all, P > 0.05). Likewise, there were no changes in the phosphorylation of p70S6K, AMPKα, or total and phosphorylated yes-associated protein on Ser127. Finally, postabsorptive MPS was lower in premenopausal versus postmenopausal females ( P = 0.023). Our results demonstrate that resistance exercise-induced changes in MPS are temporally regulated, but do not result in greater cumulative (0-4 h) MPS in postmenopausal women. NEW & NOTEWORTHY An adequate quality and quantity of skeletal muscle is relevant to support physical performance and metabolic health. Muscle protein synthesis (MPS) is an established remodeling marker, which can be hypertrophic or nonhypertrophic. Importantly, protein ingestion and resistance exercise are two strategies that support healthy muscle by stimulating MPS. Our study shows postmenopause modulates baseline MPS that may diminish the MPS response to the fundamental anabolic stimuli of protein ingestion and resistance exercise in older females.

Aquaporin 2 (AQP2) is a vasopressin (VP)-regulated water channel in the renal collecting duct. Phosphorylation and ubiquitylation of AQP2 play an essential role in controlling the cellular abundance of AQP2 and its accumulation on the plasma membrane in response to VP. Cullin-RING ubiquitin ligases (CRLs) are multisubunit E3 ligases involved in ubiquitylation and degradation of their target proteins, eight of which are expressed in the collecting duct. Here, we used an established cell model of the collecting duct (mpkCCD14 cells) to study the role of cullins in modulating AQP2. Western blotting identified Cul-1 to Cul-5 in mpkCCD14 cells. Treatment of cells for 4 h with a pan-cullin inhibitor (MLN4924) decreased AQP2 abundance, prevented a VP-induced reduction in AQP2 Ser 261 phosphorylation, and attenuated VP-induced plasma membrane accumulation of AQP2 relative to the vehicle. AQP2 ubiquitylation levels were significantly higher after MLN4924 treatment compared with controls, and they remained higher despite VP treatment. Cullin inhibition increased ERK1/2 activity, a kinase that regulates AQP2 Ser 261 phosphorylation, and VP-induced reductions in ERK1/2 phosphorylation were absent during MLN4924 treatment. Furthermore, the greater Ser 261 phosphorylation and reduction in AQP2 abundance during MLN4924 treatment were attenuated during ERK1/2 inhibition. MLN4924 increased intracellular calcium levels via calcium release-activated calcium channels, inhibition of which abolished MLN4924 effects on Ser 261 phosphorylation and AQP2 abundance. In conclusion, CRLs play a vital role in mediating some of the effects of VP to increase AQP2 plasma membrane accumulation and AQP2 abundance. Whether modulation of cullin activity can contribute to body water homeostasis requires further studies. NEW & NOTEWORTHY Aquaporin 2 (AQP2) is essential for body water homeostasis and is regulated by the antidiuretic hormone vasopressin. The posttranslational modification ubiquitylation is a key regulator of AQP2 abundance and plasma membrane localization. Here we demonstrate that cullin-RING E3 ligases play a vital role in mediating some of the effects of vasopressin to increase AQP2 abundance and plasma membrane accumulation. The results suggest that manipulating cullin activity could be a novel strategy to alter kidney water handling.

The phosphatidylinositol 3-kinase-Akt signaling pathway plays an essential role in regulating cell proliferation and apoptosis. Akt kinase is at the center of this signaling pathway and interacts with a variety of proteins. Akt is overexpressed in almost 80% of tumors. However, inhibiting Akt has serious clinical side effects so is not a suitable treatment for cancer. During recent years, Akt scaffold proteins have received increasing attention for their ability to regulate Akt signaling and have emerged as potential targets for cancer therapy. In this paper, we categorize Akt kinase scaffold proteins into four groups based on their cellular location: membrane-bound activator and inhibitor, cytoplasm, and endosome. We describe how these scaffolds interact with Akt kinase, how they affect Akt activity, and how they regulate the specificity of Akt signaling. We also discuss the clinical application of Akt scaffold proteins as targets for cancer therapy. [ABSTRACT FROM AUTHOR]

Skeletal muscle protein turnover plays a crucial role in controlling muscle mass and protein quality control, including sarcomeric (structural and contractile) proteins. Protein turnover is a dynamic and continual process of protein synthesis and degradation. The ubiquitin proteasome system (UPS) is a key degradative system for protein degradation and protein quality control in skeletal muscle. UPS-mediated protein quality control is known to be impaired in aging and diseases. Exercise is a well-recognized, nonpharmacological approach to promote muscle protein turnover rates. Over the past decades, we have acquired substantial knowledge of molecular mechanisms of muscle protein synthesis after exercise. However, there have been considerable gaps in the mechanisms of how muscle protein degradation is regulated at the molecular level. The main challenge to understand muscle protein degradation is due in part to the lack of solid stable isotope tracer methodology to measure muscle protein degradation rate. Understanding the mechanisms of UPS with the concomitant measurement of protein degradation rate in skeletal muscle will help identify novel therapeutic strategies to ameliorate impaired protein turnover and protein quality control in aging and diseases. Thus, the goal of this present review was to highlight how recent advances in the field may help improve our understanding of exercise-mediated protein degradation. We discuss 1) the emerging roles of protein phosphorylation and ubiquitylation modifications in regulating proteasome-mediated protein degradation after exercise and 2) methodological advances to measure in vivo myofibrillar protein degradation rate using stable isotope tracer methods. [ABSTRACT FROM AUTHOR]

During agonist stimulation of airway smooth muscle (ASM), agonists such as ACh induce a transient increase in cytosolic Ca2+ concentration ([Ca2+ ]cyt), which leads to a contractile response [excitation-contraction (E-C) coupling]. Previously, the sensitivity of the contractile response of ASM to elevated [Ca2+ ]cyt (Ca2+ sensitivity) was assessed as the ratio of maximum force to maximum [Ca2+ ]cyt. However, this static assessment of Ca2+ sensitivity overlooks the dynamic nature of E-C coupling in ASM. In this study, we simultaneously measured [Ca2+ ]cyt and isometric force responses to three concentrations of ACh (1, 2.6, and 10 μM). Both maximum [Ca2+ ]cyt and maximum force responses were ACh concentration dependent, but force increased disproportionately, thereby increasing static Ca2+ sensitivity. The dynamic properties of E-C coupling were assessed in several ways. The temporal delay between the onset of ACh-induced [Ca2+ ]cyt and onset force responses was not affected by ACh concentration. The rates of rise of the ACh-induced [Ca2+ ]cyt and force responses increased with increasing ACh concentration. The integral of the phase-loop plot of [Ca2+ ]cyt and force from onset to steady state also increased with increasing ACh concentration, whereas the rate of relaxation remained unchanged. Although these results suggest an ACh concentration-dependent increase in the rate of cross-bridge recruitment and in the rate of rise of [Ca2+ ]cyt, the extent of regulatory myosin light-chain (rMLC20) phosphorylation was not dependent on ACh concentration. We conclude that the dynamic properties of [Ca2+ ]cyt and force responses in ASM are dependent on ACh concentration but reflect more than changes in the extent of rMLC20 phosphorylation. [ABSTRACT FROM AUTHOR]

The dystrophin-glycoprotein complex (DGC) is a multiprotein structure required to maintain muscle fiber membrane integrity, transmit force by linking the actin cytoskeleton with the extracellular matrix, and maintain muscle homeostasis. Membrane localization of dystrophin is perturbed in muscles wasting as a consequence of cancer cachexia, tenotomy, and advanced aging, which are all associated with low level, chronic inflammation. Strategies to preserve dystrophin expression at the sarcolemma might therefore combat muscle wasting. Phosphorylation of dystrophin serine 3059 (S3059) enhances the interaction between dystrophin and b-dystroglycan. To test the contribution of amino acid phosphorylation to muscle fiber size changes, dystrophin constructs with phospho-null and phosphomimetic mutations were transfected into C2C12 muscle cells or AAV-293 cells in the presence or absence of kinase inhibitors/activators to assess effects on myotube diameter and protein function. Overexpression of a dystrophin construct with a phospho-null mutation at S3059 in vitro reduced myotube size in healthy C2C12 cells. Conversely overexpression of a phosphomimetic mutation at S3059 attenuated inflammation-induced myotube atrophy. Increased ERK activation by addition of phorbol myristate acetate (PMA) also reduced inflammation-associated myotube atrophy and increased the interaction between dystrophin and b-dystroglycan. These findings demonstrate a link between increased ERK activation, dystrophin S3059 phosphorylation, stabilization of the DGC, and the regulation of muscle fiber size. Interventions that increase dystrophin S3059 phosphorylation to promote stronger binding of dystrophin to b-dystroglycan may have therapeutic potential for attenuation of inflammation-associated muscle wasting. [ABSTRACT FROM AUTHOR]

Angiotensin II (ANG II) regulates an array of physiological and pathological responses in vascular smooth muscle cells (VSMCs) by activating ERK1/2 and phosphoinositide 3-kinase (PI3K)/Akt signaling pathways. We have demonstrated that ANG II and insulin-like growth factor-1 (IGF-1) induce the expression of early growth response protein-1 (Egr-1), a zinc finger transcription factor, which regulates the transcription of cell cycle regulatory genes network in VSMCs. We have reported that IGF-1 induces the phosphorylation of histone deacetylase 5 (HDAC5), which has been implicated in the expression of genes linked to VSMC growth and hypertrophy, via a PI3K/Akt-dependent pathway in VSMCs. However, the involvement of PI3K/Akt pathways in ANG II-induced HDAC5 phosphorylation and the contribution of HDAC5 in Egr-1 expression and hypertrophy in VSMCs remain unexplored. Here, we show that pharmacological blockade of the PI3K/Akt pathway either by wortmannin/SC66 or siRNA-induced silencing of Akt attenuated ANG II-induced HDAC5 phosphorylation and its nuclear export. Moreover, SC66 or Akt knockdown also suppressed ANG II-induced Egr-1 expression. Furthermore, pharmacological inhibition of HDAC5 by MC1568 or TMP-195 or knockdown of HDAC5 and the blockade of the nuclear export of HDAC5 by leptomycin B or KPT-330 significantly reduced ANG II-induced Egr-1 expression. In addition, depletion of either HDAC5 or Egr-1 by siRNA attenuated VSMC hypertrophy in response to ANG II. In summary, our results demonstrate that ANG II-induced HDAC5 phosphorylation and its nuclear exclusion are mediated by PI3K/Akt pathway and HDAC5 is an upstream regulator of Egr-1 expression and hypertrophy in VSMCs. [ABSTRACT FROM AUTHOR]

Aquaporins (AQPs) are water channels that facilitate transport of water across cellular membranes. AQPs are overexpressed in several cancers. Especially in breast cancer, AQP5 overexpression correlates with spread to lymph nodes and poor prognosis. Previously, we showed that AQP5 expression reduced cell-cell adhesion by reducing levels of adherens and tight-junction proteins (e.g., ZO-1, plakoglobin, and b-catenin) at the actual junctions. Here, we show that, when targeted to the plasma membrane, the AQP5 COOH-terminal tail domain regulated junctional proteins and, moreover, that AQP5 interacted with ZO-1, plakoglobin, b-catenin, and desmoglein-2, which were all reduced at junctions upon AQP5 overexpression. Thus, our data suggest that AQP5 mediates the effect on cell-cell adhesion via interactions with junctional proteins independently of AQP5-mediated water transport. AQP5 overexpression in cancers may thus contribute to carcinogenesis and cancer spread by two independent mechanisms: reduced cell-cell adhesion, a characteristic of epithelial-mesenchymal transition, and increased cell migration capacity via water transport. [ABSTRACT FROM AUTHOR]

Pathological cardiac remodeling is a leading cause of mortality in patients with diabetes. Given the glucose and lipid metabolism disorders (GLDs) in patients with diabetes, it is urgent to conduct a comprehensive study of the myocardial damage under GLDs and find key mechanisms. Apolipoprotein E knockout (ApoE-/-) mice, low-density lipoprotein receptor heterozygote (Ldlr+/-) Syrian golden hamsters, or H9C2 cells were used to construct GLDs models. GLDs significantly promoted cardiomyocyte fibrosis, apoptosis, and hypertrophy in vivo and in vitro, but inhibition of galectin-3 (Gal-3) could significantly reverse this process. Then, the signal transmission pathways were determined. It was found that GLDs considerably inhibited the phosphorylation of Akt at Thr308/Ser473, whereas the silencing of Gal-3 could reverse the inhibition of Akt activity through phosphoinositide 3-kinase-AktThr308 (PI3K-AktThr308) and AMP-activated protein kinase-mammalian target of rapamycin complex 2-AktSer473 (AMPK-mTOR2-AktSer473) pathways. Finally, the PI3K, mTOR, AMPK inhibitor, and Akt activator were used to investigate the role of pathways in regulating cardiac remodeling. Phospho-AktThr308 could mediate myocardial fibrosis, whereas myocardial apoptosis and hypertrophy were regulated by both phospho-AktThr308 and phospho-AktSer473. In conclusion, Gal-3 was an important regulatory factor in GLDs-induced cardiac remodeling, and Gal-3 could suppress the phosphorylation of Akt at different sites in mediating cardiomyocyte fibrosis, apoptosis, and hypertrophy. NEW & NOTEWORTHY Studies on the pathogenesis of diabetic cardiac remodeling are highly desired. Glucose and lipid metabolism are both disordered in diabetes. Glucose and lipid metabolism disturbances promote myocardial fibrosis, apoptosis, and hypertrophy through galectin-3. Galectin-3 promotes cardiac remodeling by inhibiting phosphorylation of AktThr308 or AktSer473. The present study finds that glucose and lipid metabolism disorders are important causes for myocardial damage and provides novel ideas for the prevention and treatment of diabetic cardiac remodeling. [ABSTRACT FROM AUTHOR]

Kidney-specific with-no-lysine kinase 1 (KS-WNK1) is an isoform of WNK1 kinase that is predominantly found in the distal convoluted tubule of the kidney. The precise physiological function of KS-WNK1 remains unclear. Some studies have suggested that it could play a role in regulating potassium renal excretion by modulating the activity of the Na + -Cl - cotransporter (NCC). However, changes in the potassium diet from normal to high failed to reveal a role for KS-WNK1, but under a normal-potassium diet, the expression of KS-WNK1 is negligible. It is only detectable when mice are exposed to a low-potassium diet. In this study, we investigated the role of KS-WNK1 in regulating potassium excretion under extreme changes in potassium intake. After following a zero-potassium diet (0KD) for 10 days, KS-WNK1 -/- mice had lower plasma levels of K + and Cl - while exhibiting higher urinary excretion of Na + , Cl - , and K + compared with KS-WNK1 +/+ mice. After 10 days of 0KD or normal-potassium diet (NKD), all mice were challenged with a high-potassium diet (HKD). Plasma K + levels markedly increased after the HKD challenge only in mice previously fed with 0KD, regardless of genotype. KSWNK1 +/+ mice adapt better to HKD challenge than KS-WNK1 -/- mice after a potassium-retaining state. The difference in the phosphorylated NCC-to-NCC ratio between KS-WNK1 +/+ and KS-WNK1 -/- mice after 0KD and HKD indicates a role for KS-WNK1 in both NCC phosphorylation and dephosphorylation. These observations show that KS-WNK1 helps the distal convoluted tubule to respond to extreme changes in potassium intake, such as those occurring in wildlife. NEW & NOTEWORTHY The findings of this study demonstrate that kidney-specific with-no-lysine kinase 1 plays a role in regulating urinary electrolyte excretion during extreme changes in potassium intake, such as those occurring in wildlife.  .

Targeting alternative exons for therapeutic gain has been achieved in a few instances and potentially could be applied more broadly. The myosin phosphatase (MP) enzyme is a critical hub upon which signals converge to regulate vessel tone. Alternative exon 24 of myosin phosphatase regulatory subunit (Mypt1 E24) is an ideal target as toggling between the two isoforms sets smooth muscle sensitivity to vasodilators such as nitric oxide (NO). This study aimed to develop a gene-based therapy to suppress splicing of Mypt1 E24 thereby switching MP enzyme to the NO-responsive isoform. CRISPR/Cas9 constructs were effective at editing of Mypt1 E24 in vitro; however, targeting of vascular smooth muscle in vivo with AAV9 was inefficient. In contrast, an octo-guanidine conjugated antisense oligonucleotide targeting the 5' splice site of Mypt1 E24 was highly efficient in vivo. It reduced the percent splicing inclusion of Mypt1 E24 from 80% to 10% in mesenteric arteries. The maximal and half-maximal effects occurred at 12.5 and 6.25 mg/kg, respectively. The effect persisted for at least 1 mo without toxicity. This highly effective splice-blocking antisense oligonucleotide could be developed as a novel therapy to reverse vascular dysfunction common to diseases such as hypertension and heart failure. NEW & NOTEWORTHY Alternative exon usage is a major driver of phenotypic diversity in all cell types including smooth muscle. However, the functional significance of most of the hundreds of thousands of alternative exons has not been defined, nor in most cases even tested. If their importance to vascular function were known these alternative exons could represent novel therapeutic targets. Here, we present injection of Vivo-morpholino splice-blocking antisense oligonucleotides as a simple, efficient, and cost-effective method for suppression of alternative exon usage in vascular smooth muscle in vivo.

Prior studies showed that epidermal growth factor (EGF) inhibits vasopressin-stimulated osmotic water permeability in the renal collecting duct. Here, we investigated the underlying mechanism. Using isolated perfused rat inner medullary collecting ducts (IMCDs), we found that the addition of EGF to the peritubular bath significantly decreased 1-deamino-8-d-arginine vasopressin (dDAVP)-stimulated water permeability, confirming prior observations. The inhibitory effect of EGF on water permeability was associated with a reduction in intracellular cAMP levels and protein kinase A (PKA) activity. Using phospho-specific antibodies and immunoblotting in IMCD suspensions, we showed that EGF significantly reduces phosphorylation of AQP2 at Ser 264 and Ser 269 . This effect was absent when 8-cpt-cAMP was used to induce AQP2 phosphorylation, suggesting that EGF's inhibitory effect was at a pre-cAMP step. Immunofluorescence labeling of microdissected IMCDs showed that EGF significantly reduced apical AQP2 abundance in the presence of dDAVP. To address what protein kinase might be responsible for Ser 269 phosphorylation, we used Bayesian analysis to integrate multiple-omic datasets. Thirteen top-ranked protein kinases were subsequently tested by in vitro phosphorylation experiments for their ability to phosphorylate AQP2 peptides using a mass spectrometry readout. The results show that the PKA catalytic-α subunit increased phosphorylation at Ser 256 , Ser 264 , and Ser 269 . None of the other kinases tested phosphorylated Ser 269 . In addition, H-89 and PKI strongly inhibited dDAVP-stimulated AQP2 phosphorylation at Ser 269 . These results indicate that EGF decreases the water permeability of the IMCD by inhibiting cAMP production, thereby inhibiting PKA and decreasing AQP2 phosphorylation at Ser 269 , a site previously shown to regulate AQP2 endocytosis. NEW & NOTEWORTHY The authors used native rat collecting ducts to show that inhibition of vasopressin-stimulated water permeability by epidermal growth factor involves a reduction of aquaporin 2 phosphorylation at Ser 269 , a consequence of reduced cAMP production and PKA activity.

During the development of pleural fibrosis, pleural mesothelial cells (PMCs) undergo phenotypic switching from differentiated mesothelial cells to mesenchymal cells (MesoMT). Here, we investigated how external stimuli such as TGF-β induce HPMC-derived myofibroblast differentiation to facilitate the development of pleural fibrosis. TGF-β significantly increased di-phosphorylation but not mono-phosphorylation of myosin II regulatory light chain (RLC) in HPMCs. An increase in RLC di-phosphorylation was also found at the pleural layer of our carbon black bleomycin (CBB) pleural fibrosis mouse model, where it showed filamentous localization that coincided with alpha smooth muscle actin (αSMA) in the cells in the pleura. Among the protein kinases that can phosphorylate myosin II RLC, ZIPK (zipper-interacting kinase) protein expression was significantly augmented after TGF-β stimulation. Furthermore, ZIPK gene silencing attenuated RLC di-phosphorylation, suggesting that ZIPK is responsible for di-phosphorylation of myosin II in HPMCs. Although TGF-β significantly increased the expression of ZIP kinase protein, the change in ZIP kinase mRNA was marginal, suggesting a posttranscriptional mechanism for the regulation of ZIP kinase expression by TGF-β. ZIPK gene knockdown (KD) also significantly reduced TGF-β-induced upregulation of αSMA expression. This finding suggests that siZIPK attenuates myofibroblast differentiation of HPMCs. siZIPK diminished TGF-β-induced contractility of HPMCs consistent with siZIPK-induced decrease in the di-phosphorylation of myosin II RLC. The present results implicate ZIPK in the regulation of the contractility of HPMC-derived myofibroblasts, phenotype switching, and myofibroblast differentiation of HPMCs. NEW & NOTEWORTHY Here, we highlight that ZIP kinase is responsible for di-phosphorylation of myosin light chain, which facilitates stress fiber formation and actomyosin-based cell contraction during mesothelial to mesenchymal transition in human pleural mesothelial cells. This transition has a significant impact on tissue remodeling and subsequent stiffness of the pleura. This study provides insight into a new therapeutic strategy for the treatment of pleural fibrosis.

Vasopressin regulates water homeostasis via the V2 receptor in the kidney at least in part through protein kinase A (PKA) activation. Vasopressin, through an unknown pathway, upregulates the activity and phosphorylation of Na + -Cl - cotransporter (NCC) and Na + -K + -2Cl - cotransporter 2 (NKCC2) by Ste20-related proline/alanine-rich kinase (SPAK) and oxidative stress-responsive kinase 1 (OSR1), which are regulated by the with-no-lysine kinase (WNK) family. Phosphorylation of WNK4 at PKA consensus motifs may be involved. Inhibitor 1 (I1), a protein phosphatase 1 (PP1) inhibitor, may also play a role. In human embryonic kidney (HEK)-293 cells, we assessed the phosphorylation of WNK4, SPAK, NCC, or NKCC2 in response to forskolin or desmopressin. WNK4 and cotransporter phosphorylation were studied in desmopressin-infused WNK4 mice and in tubule suspensions. In HEK-293 cells, only wild-type WNK4 but not WNK1, WNK3, or a WNK4 mutant lacking PKA phosphorylation motifs could upregulate SPAK or cotransporter phosphorylation in response to forskolin or desmopressin. I1 transfection maximized SPAK phosphorylation in response to forskolin in the presence of WNK4 but not of mutant WNK4 lacking PP1 regulation. We observed direct PP1 regulation of NKCC2 dephosphorylation but not of NCC or SPAK in the absence of WNK4. -/- mice with desmopressin treatment did not increase SPAK/OSR1, NCC, or NKCC2 phosphorylation. In stimulated tubule suspensions from WNK4 mice, upregulation of pNKCC2 was reduced, whereas upregulation of SPAK phosphorylation was absent. These findings suggest that WNK4 is a central node in which kinase and phosphatase signaling converge to connect cAMP signaling to the SPAK/OSR1-NCC/NKCC2 pathway. -/- With-no-lysine kinases regulate the phosphorylation and activity of the Na WNK4 -/- mice, upregulation of pNKCC2 was reduced, whereas upregulation of SPAK phosphorylation was absent. These findings suggest that WNK4 is a central node in which kinase and phosphatase signaling converge to connect cAMP signaling to the SPAK/OSR1-NCC/NKCC2 pathway. NEW & NOTEWORTHY With-no-lysine kinases regulate the phosphorylation and activity of the Na + -Cl - cotransporters. This pathway is modulated by arginine vasopressin (AVP). However, the link between AVP and WNK signaling remains unknown. Here, we show that AVP activates WNK4 through increased phosphorylation at putative protein kinase A-regulated sites and decreases its dephosphorylation by protein phosphatase 1. This work increases our understanding of the signaling pathways mediating AVP actions in the kidney.+ -K + -2Cl - cotransporters. This pathway is modulated by arginine vasopressin (AVP). However, the link between AVP and WNK signaling remains unknown. Here, we show that AVP activates WNK4 through increased phosphorylation at putative protein kinase A-regulated sites and decreases its dephosphorylation by protein phosphatase 1. This work increases our understanding of the signaling pathways mediating AVP actions in the kidney.

Tumor necrosis factor α (TNFα), a proinflammatory cytokine, plays a significant role in mediating the effects of acute inflammation in response to allergens, pollutants, and respiratory infections. Previously, we showed that acute exposure to TNFα induces mitochondrial fragmentation in human airway smooth muscle (hASM) cells, which is associated with increased expression of dynamin-related protein 1 (DRP1). Phosphorylation of DRP1 at serine 616 (pDRP1 S616 ) promotes its translocation and binding to the outer mitochondrial membrane (OMM) and mediates mitochondrial fragmentation. Previously, we reported that TNFα exposure triggers protein unfolding and triggers an endoplasmic reticulum (ER) stress response involving phosphorylation of inositol-requiring enzyme 1α (pIRE1α) at serine 724 (pIRE1α S724 ) and subsequent splicing of X-box binding protein 1 (XBP1s) in hASM cells. We hypothesize that TNFα-mediated activation of the pIRE1α S724 /XBP1s ER stress pathway in hASM cells transcriptionally activates genes that encode kinases responsible for pDRP1 S616 phosphorylation. Using 3-D confocal imaging of MitoTracker green-labeled mitochondria, we found that TNFα treatment for 6 h induces mitochondrial fragmentation in hASM cells. We also confirmed that 6 h TNFα treatment activates the pIRE1α/XBP1s ER stress pathway. Using in silico analysis and ChIP assay, we showed that CDK1 and CDK5, kinases involved in the phosphorylation of pDRP1 S616 , are transcriptionally targeted by XBP1s. TNFα treatment increased the binding affinity of XBP1s on the promoter regions of CDK1 and CDK5, and this was associated with an increase in pDRP1 S616 and mitochondria fragmentation. This study reveals a new underlying molecular mechanism for TNFα-induced mitochondrial fragmentation in hASM cells. NEW & NOTEWORTHY Airway inflammation is increasing worldwide. Proinflammatory cytokines mediate an adaptive mechanism to overcome inflammation-induced cellular stress. Previously, we reported that TNFα mediates hASM cellular responses, leading to increased force and ATP consumption associated with increased O 2 consumption, and oxidative stress. This study indicates that TNFα induces ER stress, which induces mitochondrial fragmentation via pIRE1α S724 /XBP1s mediated CDK1/5 upregulation and pDRP1 S616 phosphorylation. Mitochondrial fragmentation may promote hASM mitochondrial biogenesis to maintain healthy mitochondrial pool.

The with-no-lysine kinase 4 (WNK4)-sterile 20/SPS-1-related proline/alanine-rich kinase (SPAK)/oxidative stress-responsive kinase 1 (OSR1) pathway mediates activating phosphorylation of the furosemide-sensitive Na + -K + -2Cl - cotransporter (NKCC2) and the thiazide-sensitive NaCl cotransporter (NCC). The commonly used pT96/pT101-pNKCC2 antibody cross-reacts with pT53-NCC in mice on the C57BL/6 background due to a five amino acid deletion. We generated a new C57BL/6-specific pNKCC2 antibody (anti-pT96-NKCC2) and tested the hypothesis that the WNK4-SPAK/OSR1 pathway strongly regulates the phosphorylation of NCC but not NKCC2. In C57BL/6 mice, anti-pT96-NKCC2 detected pNKCC2 and did not cross-react with NCC. Abundances of pT96-NKCC2 and pT53-NCC were evaluated in Wnk4 -/- , Osr1 -/- , Spak -/- , and Osr1 -/- / Spak -/- mice and in several models of the disease familial hyperkalemic hypertension (FHHt) in which the CUL3-KLHL3 ubiquitin ligase complex that promotes WNK4 degradation is dysregulated ( Cul3 +/-/Δ9 , Klhl3 -/- , and Klhl3 R528H/R528H ). All mice were on the C57BL/6 background. In Wnk4 -/- and controls. pT96-NKCC2 abundance was also unchanged in the other FHHt mouse models. Our data show that disruption of the WNK4-SPAK/OSR1 pathway only mildly affects NKCC2 phosphorylation, suggesting a role for other kinases in NKCC2 activation. In FHHt models NKCC2 phosphorylation is unchanged despite higher WNK4 abundance, explaining the thiazide sensitivity of FHHt. Spak The renal cation cotransporters NCC and NKCC2 are activated following phosphorylation mediated by the WNK4-SPAK/OSR1 pathway. While disruption of this pathway strongly affects NCC activity, effects on NKCC2 activity are unclear since the commonly used phospho-NKCC2 antibody was recently reported to cross-react with phospho-NCC in mice on the C57BL/6 background. Using a new phospho-NKCC2 antibody specific for C57BL/6, we show that inhibition or activation of the WNK4-SPAK/OSR1 pathway in mice only mildly affects NKCC2 phosphorylation.-/- and Osr1 -/- / Spak -/- mice, but pT96-NKCC2 abundance did not differ from controls. pT96-NKCC2/total NKCC2 was slightly lower in Osr1 -/- and Osr1 -/- / Spak -/- mice. WNK4 expression colocalized not only with NCC but also with NKCC2 in Klhl3 -/- mice, but pT96-NKCC2 abundance was unchanged. Consistent with this, furosemide-induced urinary Na + excretion following thiazide treatment was similar between Klhl3 -/- and controls. pT96-NKCC2 abundance was also unchanged in the other FHHt mouse models. Our data show that disruption of the WNK4-SPAK/OSR1 pathway only mildly affects NKCC2 phosphorylation, suggesting a role for other kinases in NKCC2 activation. In FHHt models NKCC2 phosphorylation is unchanged despite higher WNK4 abundance, explaining the thiazide sensitivity of FHHt. NEW & NOTEWORTHY The renal cation cotransporters NCC and NKCC2 are activated following phosphorylation mediated by the WNK4-SPAK/OSR1 pathway. While disruption of this pathway strongly affects NCC activity, effects on NKCC2 activity are unclear since the commonly used phospho-NKCC2 antibody was recently reported to cross-react with phospho-NCC in mice on the C57BL/6 background. Using a new phospho-NKCC2 antibody specific for C57BL/6, we show that inhibition or activation of the WNK4-SPAK/OSR1 pathway in mice only mildly affects NKCC2 phosphorylation.

Electroneutral NaCl transport by Na + /H + exchanger 3 (NHE3, SLC9A3 ) is the major Na + absorptive mechanism in the intestine and decreased NHE3 activity contributes to diarrhea. Patients with diabetes often experience gastrointestinal adverse effects and medications are often a culprit for chronic diarrhea in type 2 diabetes (T2D). We have shown previously that metformin, the most widely prescribed drug for the treatment of T2D, induces diarrhea by inhibition of Na+/H+ exchanger 3 (NHE3) in rodent models of T2D. Metformin was shown to activate AMP-activated protein kinase (AMPK), but AMPK-independent glycemic effects of metformin are also known. The current study is undertaken to determine whether metformin inhibits NHE3 by activation of AMPK and the mechanism by which NHE3 is inhibited by AMPK. Inhibition of NHE3 by metformin was abolished by knockdown of AMPK-α1 or AMPK-α2. AMPK activation by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) phosphorylated NHE3 at S555. S555 is the primary site of phosphorylation by protein kinase A (PKA), but AMPK phosphorylated S555 independently of PKA. Using Mass spectrometry, we found S563 as a newly recognized phosphorylation site in NHE3. Altering either S555 or S563 to Ala was sufficient to block the inhibition of NHE3 activity by AMPK. NHE3 inhibition is dependent on ubiquitination by the E3 ubiquitin ligase Nedd4-2 and metformin was shown to induce NHE3 internalization via Nedd4-2-mediated ubiquitination. AICAR did not increase NHE3 ubiquitination when S555 or S563 was mutated. We conclude that AMPK activation inhibits NHE3 activity and NHE3 inhibition is associated with phosphorylation of NHE3 at S555 and S563. NEW & NOTEWORTHY We show that AMP-activated protein kinase (AMPK) phosphorylates NHE3 at S555 and S563 to inhibit NHE3 activity in intestinal epithelial cells. Phosphorylation of NHE3 by AMPK is necessary for ubiquitination of NHE3.

Vasopressin regulates osmotic water transport in the renal collecting duct by protein kinase A (PKA)-mediated control of the water channel aquaporin-2 (AQP2). Collecting duct principal cells express two seemingly redundant PKA catalytic subunits, PKA catalytic α (PKA-Cα) and PKA catalytic β (PKA-Cβ). To identify the roles of these two protein kinases, we carried out deep phosphoproteomic analysis in cultured mpkCCD cells in which either PKA-Cα or PKA-Cβ was deleted using CRISPR-Cas9-based genome editing. Controls were cells carried through the genome editing procedure but without deletion of PKA. TMT mass tagging was used for protein mass spectrometric quantification. Of the 4,635 phosphopeptides that were quantified, 67 phosphopeptides were significantly altered in abundance with PKA-Cα deletion, whereas 21 phosphopeptides were significantly altered in abundance with PKA-Cβ deletion. However, only four sites were changed in both. The target proteins identified in PKA-Cα-null cells were largely associated with cell membranes and membrane vesicles, whereas target proteins in PKA-Cβ-null cells were largely associated with the actin cytoskeleton and cell junctions. In contrast, in vitro incubation of mpkCCD proteins with recombinant PKA-Cα and PKA-Cβ resulted in virtually identical phosphorylation changes. In addition, analysis of total protein abundances in in vivo samples showed that PKA-Cα deletion resulted in a near disappearance of AQP2 protein, whereas PKA-Cβ deletion did not decrease AQP2 abundance. We conclude that PKA-Cα and PKA-Cβ serve substantially different regulatory functions in renal collecting duct cells and that differences in phosphorylation targets may be due to differences in protein interactions, e.g., mediated by A-kinase anchor proteins, C-kinase anchoring proteins, or PDZ binding. [ABSTRACT FROM AUTHOR]

G551D is a major disease-associated gating mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) protein, an ATP- and phosphorylation-dependent chloride channel. G551D causes severe cystic fibrosis (CF) disease by disrupting ATP-dependent channel opening; however, whether G551D affects phosphorylation-dependent channel activation is unclear. Here, we use macropatch recording and Ussing chamber approaches to demonstrate that G551D impacts on phosphorylation-dependent activation of CFTR, and PKA-mediated phosphorylation regulates the interaction between the x-loop in nucleotide-binding domain 2 (NBD2) and cytosolic loop (CL) 1. We show that G551D not only disrupts ATP-dependent channel opening but also impairs phosphorylation-dependent channel activation by largely reducing PKA sensitivity consistent with the reciprocal relationship between channel opening/gating, ligand binding, and phosphorylation. Furthermore, we identified two novel GOF mutations: D1341R in the x-loop near the ATP-binding cassette signature motif in NBD2 and D173R in CL1, each of which strongly increased PKA sensitivity both in the wild-type (WT) background and when introduced into G551D-CFTR. When D1341R was combined with a second GOF mutation (e.g., K978C in CL3), we find that the double GOF mutation maximally increased G551D channel activity such that VX-770 had no further effect. We further show that a double chargereversal mutation of D1341R/D173R-CFTR exhibited similar PKA sensitivity when compared with WT-CFTR. Together, our results suggest that charge repulsion between D173 and D1341 of WT-CFTR normally inhibits channel activation at low PKA activity by reducing PKA sensitivity, and negative allostery by the G551D is coupled to reduced PKA sensitivity of CFTR that can be restored by second GOF mutations. [ABSTRACT FROM AUTHOR]

Aquaporin-2 (AQP2) is a vasopressin-regulated water channel protein responsible for water reabsorption by the kidney collecting ducts. Under control conditions, most AQP2 resides in the recycling endosomes of principal cells, where it answers to vasopressin with trafficking to the apical plasma membrane to increase water reabsorption. Upon vasopressin withdrawal, apical AQP2 retreats to the early endosomes before joining the recycling endosomes for the next vasopressin stimulation. Prior studies have demonstrated a role of AQP2 S269 phosphorylation in reducing AQP2 endocytosis, thereby prolonging apical AQP2 retention. Here, we studied where in the cells S269 was phosphorylated and dephosphorylated in response to vasopressin versus withdrawal. In mpkCCD collecting cells, vacuolar protein sorting 35 knockdown slowed vasopressin- induced apical AQP2 trafficking, resulting in AQP2 accumulation in the recycling endosomes where S269 was phosphorylated. Rab7 knockdown, which impaired AQP2 trafficking from the early to recycling endosomes, reduced vasopressin-induced S269 phosphorylation. Rab5 knockdown, which impaired AQP2 endocytosis, did not affect vasopressin-induced S269 phosphorylation. Upon vasopressin withdrawal, S269 was not dephosphorylated in Rab5 knockdown cells. In contrast, S269 dephosphorylation upon vasopressin withdrawal was completed in Rab7 or vacuolar protein sorting 35 knockdown cells. We conclude that S269 is dephosphorylated during Rab5-mediated AQP2 endocytosis before AQP2 joins the recycling endosomes upon vasopressin withdrawal. While in the recycling endosomes, AQP2 can be phosphorylated at S269 in response to vasopressin before apical trafficking. [ABSTRACT FROM AUTHOR]

The function of site-specific phosphorylation of nucleophosmin (NPM), an essential Bax chaperone, in stress-induced cell death is unknown. We hypothesized that NPM threonine 95 (T95) phosphorylation both signals and promotes cell death. In resting cells, NPM exclusively resides in the nucleus and T95 is nonphosphorylated. In contrast, phosphorylated T95 NPM (pNPM T95) accumulates in the cytosol after metabolic stress, in multiple human cancer cell lines following γ-radiation, and in postischemic human kidney tissue. Based on the T95 phosphorylation consensus sequence, we hypothesized that glycogen synthase kinase-3α (GSK-3α) regulates cytosolic NPM translocation by phosphorylating T95 NPM. In a cell-free system, GSK-3α phosphorylated a synthetic NPM peptide containing T95. In vitro, bidirectional manipulation of GSK-3α activity substantially altered T95 phosphorylation, cytosolic NPM translocation, and cell survival during stress, mechanistically linking these lethal events. Furthermore, GSK-3α inhibition in vivo decreased cytosolic pNPM T95 accumulation in kidney tissue after experimental ischemia. In patients with acute kidney injury, both cytosolic NPM accumulation in proximal tubule cells and NPM-rich intratubular casts were detected in frozen renal biopsy tissue. These observations show, for the first time, that GSK-3α promotes cell death partly by phosphorylating NPM at T95, to promote cytosolic NPM accumulation. T95 NPM is also a rational therapeutic target to ameliorate ischemic renal cell injury and may be a universal injury marker in mammalian cells. [ABSTRACT FROM AUTHOR]

Podocyte dysfunction contributes to proteinuric chronic kidney disease. A number of key proteins are essential for podocyte function, including nephrin, podocin, CD2- associated protein (CD2AP), synaptopodin, and -actinin-4 (ACTN4). Although most of these proteins were first identified through genetic studies associated with human kidney disease, subsequent studies have identified phosphorylation of these proteins as an important posttranslational event that regulates their function. In this review, a brief overview of the function of these key podocyte proteins is provided. Second, the role of phosphorylation in regulating the function of these proteins is described. Third, the association between these phosphorylation pathways and kidney disease is reviewed. Finally, challenges and future directions in studying phosphorylation are discussed. Better characterization of these phosphorylation pathways and others yet to be discovered holds promise for translating this knowledge into new therapies for patients with proteinuric chronic kidney disease. [ABSTRACT FROM AUTHOR]

Dysregulation of the mechanical properties and cell adhesive interactions of trabecular meshwork (TM) are known to impair aqueous humor drainage and elevate intraocular pressure in glaucoma patients. The identity of regulatory mechanisms underlying TM mechanotransduction, however, remains elusive. Here we analyzed the phosphotyrosine proteome of human TM cell-extracellular matrix (ECM) adhesion complexes, which play a key role in sensing and transducing extracellular chemical and mechanical cues into intracellular activities, using a two-level affinity pull-down (phosphotyrosine antibody and titanium dioxide beads) method and mass spectrometry. This analysis identified ~1,000 tyrosine-phosphorylated proteins of TM cell-ECM adhesion complexes. Many consensus adhesome proteins were found to be tyrosine phosphorylated. Interestingly, several of the phosphotyrosinylated proteins found in TM cell-ECM adhesion complexes are known to be required for podocyte glomerular filtration, indicating the existence of molecular parallels that are likely relevant to the shared fluid barrier and filtration functions of the two mechanosensitive cell types. [ABSTRACT FROM AUTHOR]

Skeletal muscle is a target of contraction-induced loading (CiL), leading to protein unfolding or cellular perturbations, respectively. While cytoskeletal desmin is responsible for ongoing structural stabilization, in the immediate response to CiL, alpha-crystallin B (CRYAB) is phosphorylated at serine 59 (pCRYABS59) by P38, acutely protecting the cytoskeleton. To reveal adaptation and deadaptation of these myofibrillar subsystems to CiL, we examined CRYAB, P38, and desmin regulation following resistance exercise at diverse time points of a chronic training period. Mechanosensitive JNK phosphorylation (pJNKT183/Y185) was determined to indicate the presence of mechanical components in CiL. Within 6 wk, subjects performed 13 resistance exercise bouts at the 8-12 repetition maximum, followed by 10 days detraining and a final 14th bout. Biopsies were taken at baseline and after the 1st, 3rd, 7th, 10th, 13th, and 14th bout. To assess whether potential desensitization to CiL can be mitigated, one group trained with progressive and a second with constant loading. As no group differences were found, all subjects were combined for statistics. Total and phosphorylated P38 was not regulated over the time course. pCRYABS59 and and pJNKT183/Y185 strongly increased following the unaccustomed first bout. This exercise-induced pCRYABS59/pJNKT183/Y185 increase disappeared with the 10th until 13th bout. As response to the detraining period, the 14th bout led to a renewed increase in pCRYABS59. Desmin content followed pCRYABS59 inversely, i.e., was up- when pCRYABS59 was downregulated and vice versa. In conclusion, the pCRYABS59 response indicates increase and decrease in resistance to CiL, in which a reinforced desmin network could play an essential role by structurally stabilizing the cells. [ABSTRACT FROM AUTHOR]

In mammals, conservation of body water is critical for survival and is dependent on the kidneys' ability to minimize water loss in the urine during periods of water deprivation. The collecting duct water channel aquaporin-2 (AQP2) plays an essential role in this homeostatic response by facilitating water reabsorption along osmotic gradients. The ability to increase the levels of AQP2 in the apical plasma membrane following an increase in plasma osmolality is a rate-limiting step in water reabsorption, a process that is tightly regulated by the antidiuretic hormone arginine vasopressin (AVP). In this review, the focus is on the role of the carboxyl-terminus of AQP2 as a key regulatory point for AQP2 trafficking. We provide an overview of AQP2 structure, disease-causing mutations in the AQP2 carboxyl-terminus, the role of posttranslational modifications such as phosphorylation and ubiquitylation in the tail domain, and their implications for balanced trafficking of AQP2. Finally, we discuss how various modifications of the AQP2 tail facilitate selective protein-protein interactions that modulate the AQP2 trafficking mechanism. [ABSTRACT FROM AUTHOR]

The β1-adrenergic regulation of cardiac myocyte contraction plays an important role in regulating heart function. Activation of this system leads to an increased heart rate and stronger myocyte contraction. However, chronic stimulation of the β1-adrenergic signaling system can lead to cardiac hypertrophy and heart failure. To understand the mechanisms of action of β1-adrenoceptors, a mathematical model of cardiac myocyte contraction that includes the β1-adrenergic system was developed and studied. The model was able to simulate major experimental protocols for measurements of steady-state force-calcium relationships, cross-bridge release rate and force development rate, force-velocity relationship, and force redevelopment rate. It also reproduced quite well frequency and isoproterenol dependencies for intracellular Ca2+ concentration ([Ca2+]i) transients, total contraction force, and sarcomere shortening. The mathematical model suggested the mechanisms of increased contraction force and myocyte shortening on stimulation of β1-adrenergic receptors is due to phosphorylation of troponin I and myosin-binding protein C and increased [Ca2+]i transient resulting from activation of the β1-adrenergic signaling system. The model was used to simulate work-loop contractions and estimate the power during the cardiac cycle as well as the effects of 4-aminopyridine and tedisamil on the myocyte contraction. The developed mathematical model can be used further for simulations of contraction of ventricular myocytes from genetically modified mice and myocytes from mice with chronic cardiac diseases. NEW & NOTEWORTHY A new mathematical model of mouse ventricular myocyte contraction that includes the β1-adrenergic system was developed. The model simulated major experimental protocols for myocyte contraction and predicted the effects of 4-aminopyridine and tedisamil on the myocyte contraction. The model also allowed for simulations of work-loop contractions and estimation of the power during the cardiac cycle. [ABSTRACT FROM AUTHOR]