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Simple Summary: Aerobic glycolysis is a key driving force of tumorigenesis and chemoresistance. Girdin plays a vital role in cancer cells; however, the role of Girdin in aerobic glycolysis is still unclear. In this study, we first found that knockout of Girdin markedly inhibited lung adenocarcinoma (LUAD) progression in an autochthonous LUAD mouse model. In addition, we found that Girdin interacted with pyruvate kinase M2 (PKM2) and impaired PKM2 activity, which promoted the Warburg effect and chemoresistance. Our results suggest that Girdin is a potential therapeutic target to overcome the resistance of LUAD cells to chemotherapeutic agents. Girdin, an Akt substrate, has been reported to promote tumorigenesis in various tumors. However, the role of Girdin in a spontaneous tumor model has not yet been explored. Here, we studied the role of Girdin in lung adenocarcinoma (LUAD) using the autochthonous mouse model and found that Girdin led to LUAD progression and chemoresistance by enhancing the Warburg effect. Mechanistically, Girdin interacted with pyruvate kinase M2 (PKM2), which played a vital role in aerobic glycolysis. Furthermore, Girdin impaired Platelet Derived Growth Factor Receptor Beta (PDGFRβ) degradation, which in turn, promoted PKM2 tyrosine residue 105 (Y105) phosphorylation and inhibited PKM2 activity, subsequently promoting aerobic glycolysis in cancer cells. Taken together, our study demonstrates that Girdin is a crucial regulator of tumor growth and may be a potential therapeutic target for overcoming the resistance of LUAD cells to chemotherapy. [ABSTRACT FROM AUTHOR]

Amino acid signaling mediated by the activation of mechanistic target of rapamycin complex 1 (mTORC1) is fundamental to cell growth and metabolism. However, how cells negatively regulate amino acid signaling remains largely unknown. Here, we show that interaction between 4F2 heavy chain (4F2hc), a subunit of multiple amino acid transporters, and the multifunctional hub protein girders of actin filaments (Girdin) down-regulates mTORC1 activity. 4F2hc interacts with Girdin in mitogen-activated protein kinase (MAPK)- and amino acid signaling–dependent manners to translocate to the lysosome. The resultant decrease in cell surface 4F2hc leads to lowered cytoplasmic glutamine (Gln) and leucine (Leu) content, which down-regulates amino acid signaling. Consistently, Girdin depletion augments amino acid-induced mTORC1 activation and inhibits amino acid deprivation–induced autophagy. These findings uncovered the mechanism underlying negative regulation of amino acid signaling, which may play a role in tightly regulated cell growth and metabolism. [ABSTRACT FROM AUTHOR]

Summary: Tuft cells (TCs) are minor components of gastrointestinal epithelia, characterized by apical tufts and spool-shaped somas. The lack of reliable TC-markers has hindered the elucidation of its role. We developed site-specific and phosphorylation-status–specific antibodies against Girdin at tyrosine-1798 (pY1798) and found pY1798 immunostaining of mouse jejunum clearly depicted epithelial cells closely resembling TCs. This study aimed to validate pY1798 as a TC-marker. Double-fluorescence staining of intestines was performed with pY1798 and known TC-markers, for example, hematopoietic-prostaglandin-D-synthase (HPGDS), or doublecortin-like kinase 1 (DCLK1). Odds ratios (ORs) were calculated from cell counts to determine whether two markers were attracting (OR<1) or repelling (OR>1). In consequence, pY1798 signals strongly attracted those of known TC-markers. ORs for HPGDS in mouse stomach, small intestine, and colon were 0 for all, and 0.08 for DCLK1 in human small intestine. pY1798-positive cells in jejunum were distinct from other minor epithelial cells, including goblet, Paneth, and neuroendocrine cells. Thus, pY1798 was validated as a TC-marker. Interestingly, apoptosis inducers significantly increased relative TC frequencies despite the absence of proliferation at baseline. In conclusion, pY1798 is a novel TC-marker. Selective tyrosine phosphorylation and possible resistance to apoptosis inducers implied the activation of certain kinase(s) in TCs, which may become a clue to elucidate the enigmatic roles of TCs. [ABSTRACT FROM AUTHOR]

Centralspindlin, which is composed of MgcRacGAP and MKLP1, is essential for central spindle formation and cytokinetic furrow ingression. MgcRacGAP utilizes its GAP domain to inactivate Rac1 and induce furrow ingression in mammalian cells. In this report, we present a novel regulatory mechanism for furrowing that is mediated by the phosphorylation of SHC SH2-domain binding protein 1 (SHCBP1), a binding partner of centralspindlin, by Aurora B (AurB). AurB phosphorylates Ser634 of SHCBP1 during mitosis. We generated a phosphorylation site mutant, S634A-SHCBP1, which was prematurely recruited to the central spindle during anaphase and inhibited furrowing. An in vitro GAP assay demonstrated that SHCBP1 can suppress the MgcRacGAP-mediated inactivation of Rac1. In addition, the inhibition of Rac1 activity rescued the furrowing defect induced by S634A-SHCBP1 expression. Thus, AurB phosphorylates SHCBP1 to prevent the premature localization of SHCBP1 to the central spindle and ensures that MgcRacGAP inactivates Rac1 to promote the ingression of the cytokinetic furrow. [ABSTRACT FROM AUTHOR]

Phosphorylation of actin-binding proteins plays a pivotal role in the remodeling of the actin cytoskeleton to regulate cell migration. Palladin is an actin-binding protein that is phosphorylated by growth factor stimulation; however, the identity of the involved protein kinases remains elusive. In this study, we report that palladin is a novel substrate of extracellular signalregulated kinase (ERK). Suppression of ERK activation by a chemical inhibitor reduced palladin phosphorylation, and expression of active MEK alone was sufficient for phosphorylation. In addition, an in vitro kinase assay demonstrated direct palladin phosphorylation by ERK. We found that Ser77 and Ser197 are essential residues for phosphorylation. Although the phosphorylation of these residues was not required for actin cytoskeletal organization, we found that expression of nonphosphorylated palladin enhanced cell migration. Finally, we show that phosphorylation inhibits the palladin association with Abl tyrosine kinase. Taken together, our results indicate that palladin phosphorylation by ERK has an anti-migratory function, possibly by modulating interactions with molecules that regulate cell migration. [ABSTRACT FROM AUTHOR]

It is well known that the cell cycle is controlled by several cyclin/cyclin-dependent kinase (Cdk) complexes whose expression and phosphorylation states vary with orderly periodicity. During the cell cycle, activity of the cyclin/Cdk complexes can be regulated directly or indirectly by a number of molecules, including protein kinases and phosphatases, p53, and Cdk inhibitors. Here, we show that the addition of glial cell line-derived neurotrophic factor (GDNF) induced G2/M cell cycle delay in human SK-N-MC neuroectodermal tumor cells that express RET tyrosine kinase, accompanying actin reorganization. Cell cycle delay at G2/M was characterized by accelerated and prolonged Cdc2 phosphorylation and stabilization of cyclin B1 and Wee1 kinase expression. Interestingly, we found that phosphorylation and/or expression of Cdc2, cyclinB1, and Wee1 was controlled by the Rac1/c-Jun NH2-terminal kinase (JNK) pathway. Immunohistochemical analysis suggested that the G2/M cell cycle delay may be necessary to prevent the mitotic progression of SK-N-MC cells with perturbed actin cytoskeletons. [ABSTRACT FROM AUTHOR]

Glial cell line-derived neurotrophic factor (GDNF) signals through a unique receptor system that includes Ret receptor tyrosine kinase and a glycosyl-phosphatidylinositol-linked cell surface protein. In the present study, we have identified several proteins in neuroblastoma cells that are phosphorylated on tyrosine in response to GDNF. The phosphorylated proteins include focal adhesion kinase (FAK), paxillin and Crk-associated substrate, p130Cas, all of which are known to be associated with focal adhesions. Of these, paxillin and p130Cas interacted with Crk proteins in GDNF-treated neuroblastoma cells. GDNF also induced reorganization of the actin cytoskelton. Tyrosine phosphorylation of FAK, paxillin and p130Cas was inhibited by cytochalasin D or two specific inhibitors of phosphatidylinositol-3′ kinase (PI-3′ kinase), wortmannin and LY294002, indicating that their tyrosine phosphorylation depends on the formation of actin stress fiber and activation of PI-3′ kinase. In addition, phosphorylation of FAK but not of paxillin and p130Cas was markedly impaired by the Clostridium botulinum C3 exoenzyme that specifically ADP-ribosylates and inactivates Rho. These results suggested the presence of Rho-dependent and -independent signaling pathways downstream of PI-3′ kinase that mediate tyrosine phosphorylation of FAK, paxillin and p130Cas through Ret kinase. [ABSTRACT FROM AUTHOR]

The canonical Wnt signaling pathway plays a crucial role in embryonic development, tissue homeostasis and cancer progression. The binding of Wnt ligands to their cognate receptors, the Frizzled (Fzd) family of proteins, recruits Dishevelled segment polarity protein (Dvl) to the plasma membrane and induces its phosphorylation via casein kinase 1 (CK1), which leads to the activation of β-catenin. Previous studies showed that Dishevelled-associating protein with a high frequency of leucine residues (Daple) is an important component of the Wnt signaling pathway and essential for Dvl phosphorylation. However, the mechanism by which Daple promotes CK1-mediated phosphorylation of Dvl is not fully understood. In this study, we found that Daple overexpression induced CK1ε-mediated Dvl2 phosphorylation at threonine 224 (Thr224). A Daple mutant (Daple ΔGCV) that lacks a carboxyl-terminal motif to associate with Dvl, retained the ability to interact with CK1ε, but did not induce Dvl phosphorylation, suggesting the importance of the Daple/Dvl/CK1ε trimeric protein complex. We further found that Thr224 phosphorylation of Dvl was required for full activation of β-catenin transcriptional activity. Consistent with this, wild-type Daple promoted β-catenin transcriptional activity, following dissociation of β-catenin and axin. Finally, Wnt3a stimulation increased the membrane localization of Daple and its association with Dvl, and Daple knockdown attenuated Wnt3a-mediated β-catenin transcriptional activity. Collectively, these data suggested a essential role of spatial Daple localization in CK1ε-mediated activation of Dvl in the canonical Wnt signaling pathway. • Dvl2 phosphorylation is essential for the activation of canonical Wnt signaling. • Daple regulates Dvl2 phosphorylation at Thr224 mediated by CK1ε. • Wnt3a ligand promotes Dvl2 phosphorylation regulated by Daple. • Daple is recruited to the membrane and robustly interacts with Dvl2 in cells stimulated with Wnt3a. [ABSTRACT FROM AUTHOR]

Myocardial infarction is a leading cause of death, and cardiac rupture following myocardial infarction leads to extremely poor prognostic feature. A large body of evidence suggests that Akt is involved in several cardiac diseases. We previously reported that Akt-mediated Girdin phosphorylation is essential for angiogenesis and neointima formation. The role of Girdin expression and phosphorylation in myocardial infarction, however, is not understood. Therefore, we employed Girdin-deficient mice and Girdin S1416A knock-in (Girdin SA/SA ) mice, replacing the Akt phosphorylation site with alanine, to address this question. We found that Girdin was expressed and phosphorylated in cardiac fibroblasts in vitro and that its phosphorylation was crucial for the proliferation and migration of cardiac fibroblasts. In vivo , Girdin was localized in non-cardiomyocyte interstitial cells and phosphorylated in α-smooth muscle actin-positive cells, which are likely to be cardiac myofibroblasts. In an acute myocardial infarction model, Girdin SA/SA suppressed the accumulation and proliferation of cardiac myofibroblasts in the infarcted area. Furthermore, lower collagen deposition in Girdin SA/SA mice impaired cardiac repair and resulted in increased mortality attributed to cardiac rupture. These findings suggest an important role of Girdin phosphorylation at serine 1416 in cardiac repair after acute myocardial infarction and provide insights into the complex mechanism of cardiac rupture through the Akt/Girdin-mediated regulation of cardiac myofibroblasts. [ABSTRACT FROM AUTHOR]

The mammalian protein Girdin interacts with several key molecules such as actin, and it functions as a regulator of the cytoskeleton. Silencing of Girdin mRNA results in defective migration in a variety of cultured cells. Moreover, knockout of Girdin causes phenotypes related to defective migration, including hypoplasia of olfactory bulbs and a widened rostral migratory stream (RMS) in mice. To elucidate the molecular basis underlying cellular migration, we generated site- and phosphorylation state-specific antibodies against human Girdin peptides carrying four putative phosphorylation sites (serine1386 [S1386], S1416, tyrosine1764 [Y1764] and Y1798) that had been identified by mutagenesis analyses or mass spectrometric studies. We found that these residues were phosphorylated in an epidermal growth factor (EGF)-dependent manner. Among the four antibodies we developed, the antibody that targeted Girdin when phosphorylated at Y1798 (pY1798) worked well for immunohistochemistry of paraffin-embedded tissues as well as for cultured cells. Immunocytochemistry of HEK293FT cells transfected with an EGF receptor expression plasmid exhibited punctate signals with pY1798. These signals colocalized with those of endocytosed EGF receptors after EGF stimulation. Signals from pY1798 were also observed on lamellipodia, filopodia, focal adhesion and stress fibers in NIH3T3 cells under conventional culture conditions. Immunohistochemistry of paraffin-embedded mouse brain at P14 using anti-pY1798 antibody displayed signals at the hilum-side (internal side) of the dentate gyrus of the hippocampus, the RMS, the accessory olfactory bulb and the olfactory bulb in which Girdin expression was detected. Primary culture of RMS neurons showed punctate signals of pY1798 at the tips of leading processes as well as in the cytoplasm, whereas no signals were observed when neurons were treated with Src inhibitor, PP2. Our data revealed the changes in the phosphorylation status of Y1798 in Girdin when it associated with migration-related structures in vitro and in vivo . [ABSTRACT FROM AUTHOR]

The catalytic and signaling activities of RET, a receptor-type tyrosine kinase, are regulated by the autophosphorylation of several tyrosine residues in the cytoplasmic region of RET. Some studies have revealed a few possible autophosphorylation sites of RET by [32P]phosphopeptide mapping or by using specific anti-phosphotyrosine antibodies. To ultimately identify these and other autophosphorylation sites of RET, we performed mass spectrometry analysis of an originally prepared RET recombinant protein. Both the autophosphorylation and kinase activity of myelin basic protein as an external substrate of the recombinant RET protein were substantially elevated in the presence of ATP without stimulation by a glial cell line-derived neurotrophic factor, a natural ligand for RET. Mass spectrometric analysis revealed that RET Tyr806, Tyr809, Tyr900, Tyr905, Tyr981, Tyr1062, Tyr1090, and Tyr1096were autophosphorylation sites. Levels of autophosphorylation and kinase activity of RET-MEN2A (multiple endocrine neoplasia 2A), a constitutively active form of RET with substitution of Tyr900 by phenylalanine (Y900F), were comparable with those of original RET-MEN2A, whereas those of the mutant Y905F were greatly decreased. Interestingly, those of a double mutant, Y900F/Y905F, were completely abolished. Both the kinase activity and transforming activity were impaired in the mutants Y806F and Y809F. These results provide convincing evidence for both previously suggested and new tyrosine autophosphorylation sites of RET as well as for novel functions of Tyr806, Tyr809, and Tyr900 phosphorylation in both catalytic kinase activities and cell growth. The significance of the identified autophosphorylation sites in various protein-tyrosine kinases registered in a data base is discussed in this paper. [ABSTRACT FROM AUTHOR]

By use of a specifically sulfhydryl group-reactive chemical, 1,4-butanediyl-bismethanethiosulfonate (BMTS), we studied the localization of oxidative stress-responsive target cysteines for activation of a receptor-type protein tyrosine kinase, c-RET. The chemical, which reacted with RET proteins on the cell surface for sulfhydryl-linked aggregation, induced autophosphorylation and activation of RET kinase. When extracellular domain-deleted RET mutant (RET-PTC-1) cells were exposed to BMTS, neither the molecular status nor the activity of the enzyme was affected, suggesting that the target cysteines of BMTS to which cells were exposed for reaction are located in the cysteine-rich region of the extracellular domain of RET kinase. Despite this result, the exposure of a subcellular form of c-RET or RET-PTC-1 kinase isolated by immunoprecipitation to BMTS did induce activation of the enzyme. These results suggest that cysteines in both the extracellular and the intracellular domains of RET can work as target sites of accessible BMTS and possibly other oxidative elements for structural modification and activation of RET kinase. [Copyright &y& Elsevier]

Abstract: c-Ret has been shown to be crucial for neural development and survival. We have recently shown that complete impairment of tyrosine 1062 (Y1062)-phosphorylation in c-Ret causes congenital hearing loss with neurodegeneration of spiral ganglion neurons (SGNs) in homozygous c-Ret knockin mice (c-Ret-KIY1062F/Y1062F-mice). However, there is no information to link c-Ret and age-related hearing loss. Here we show that partial impairment of Y1062-phosphorylation in c-Ret accelerates age-related hearing loss in heterozygous c-Ret Y1062F knockin mice (c-Ret-KIY1062F/+-mice). In contrast, complete impairment of serine 697 (S697)-phosphorylation in c-Ret did not affect hearing levels in 10-month-old homozygous c-Ret S697A knockin mice (c-Ret-KIS697A/S697A-mice). The hearing loss involved late-onset neurodegeneration of spiral ganglion neurons in c-Ret-KIY1062F/+-mice. Morphological abnormalities in inner- and outer-hair cells and the stria vascularis in c-Ret-KIY1062F/+-mice were undetectable. The acceleration of age-related hearing loss in c-Ret-KIY1062F/+-mice was rescued by introducing constitutively activated RET. Thus, our results suggest that c-Ret is a novel age-related hearing loss-related molecule in mice. Our results suggest that these hearing losses partially share a common pathogenesis that is monogenetically caused by a single point mutation (Y1062F) in c-Ret. [Copyright &y& Elsevier]