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Cancer cells undergo metabolic reprogramming that is intricately linked to malignancy. Protein acylations are especially responsive to metabolic changes, influencing signal transduction pathways and fostering cell proliferation. However, as a novel type of acylations, the involvement of malonylation in cancer remains poorly understood. In this study, we observed a significant reduction in malonyl-CoA levels in hepatocellular carcinoma (HCC), which correlated with a global decrease in malonylation. Subsequent nuclear malonylome analysis unveiled nucleolin (NCL) malonylation, which was notably enhanced in HCC biopsies. we demonstrated that NCL undergoes malonylation at lysine residues 124 and 398. This modification triggers the translocation of NCL from the nucleolus to nucleoplasm and cytoplasm, binding to AKT mRNA, and promoting AKT translation in HCC. Silencing AKT expression markedly attenuated HCC cell proliferation driven by NCL malonylation. These findings collectively highlight nuclear signaling in modulating AKT expression, suggesting NCL malonylation as a novel mechanism through which cancer cells drive cell proliferation., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Damage to the genetic material of the cell poses a universal threat to all forms of life. The DNA damage response is a coordinated cellular response to a DNA break, key to which is the phosphorylation signaling cascade. Identifying which proteins are phosphorylated is therefore crucial to understanding the mechanisms that underlie it. We have used stable isotopic labeling of amino acids in cell culture-based quantitative phosphoproteomics to profile changes in phosphorylation site abundance following double stranded DNA breaks, at two distinct loci in the genome of the single cell eukaryote Trypanosoma brucei. Here, we report on the T. brucei phosphoproteome following a single double-strand break at either a chromosome internal or subtelomeric locus, specifically the bloodstream form expression site. We detected >6500 phosphorylation sites, of which 211 form a core set of double-strand break responsive phosphorylation sites. Along with phosphorylation of canonical DNA damage factors, we have identified two novel phosphorylation events on histone H2A and found that in response to a chromosome internal break, proteins are predominantly phosphorylated, while a greater proportion of proteins dephosphorylated following a DNA break at a subtelomeric bloodstream form expression site. Our data represent the first DNA damage phosphoproteome and provides novel insights into repair at distinct chromosomal contexts in T. brucei., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a lipid-enveloped virus that acquires its lipid bilayer from the host cell it infects. SARS-CoV-2 can spread from cell to cell or from patient to patient by undergoing assembly and budding to form new virions. The assembly and budding of SARS-CoV-2 is mediated by several structural proteins known as envelope (E), membrane (M), nucleoprotein (N), and spike (S), which can form virus-like particles (VLPs) when co-expressed in mammalian cells. Assembly and budding of SARS-CoV-2 from the host ER-Golgi intermediate compartment is a critical step in the virus acquiring its lipid bilayer. To date, little information is available on how SARS-CoV-2 assembles and forms new viral particles from host membranes. In this study, we used several lipid binding assays and found the N protein can strongly associate with anionic lipids including phosphoinositides and phosphatidylserine. Moreover, we show lipid binding occurs in the N protein C-terminal domain, which is supported by extensive in silico analysis. We demonstrate anionic lipid binding occurs for both the free and the N oligomeric forms, suggesting N can associate with membranes in the nucleocapsid form. Based on these results, we present a lipid-dependent model based on in vitro, cellular, and in silico data for the recruitment of N to assembly sites in the lifecycle of SARS-CoV-2., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The Hippo-YAP signaling pathway plays a central role in many biological processes such as regulating cell fate, organ size, and tissue growth, and its key components are spatiotemporally expressed and posttranslationally modified during these processes. Neddylation is a posttranslational modification that involves the covalent attachment of NEDD8 to target proteins by NEDD8-specific E1-E2-E3 enzymes. Whether neddylation is involved in Hippo-YAP signaling remains poorly understood. Here, we provide evidence supporting the critical role of NEDD8 in facilitating the Hippo-YAP signaling pathway by mediating neddylation of the transcriptional coactivator yes-associated protein 1 (YAP1). Overexpression of NEDD8 induces YAP1 neddylation and enhances YAP1 transactivity, but inhibition of neddylation suppresses YAP1 transactivity and attenuates YAP1 nuclear accumulation. Furthermore, inhibition of YAP1 signaling promotes MLN4924-induced ovarian granulosa cells apoptosis and disruption of nedd8 in zebrafish results in downregulation of yap1-activated genes and upregulation of yap1-repressed genes. Further assays show that the xiap ligase promotes nedd8 conjugates to yap1 and that yap1 neddylation. In addition, we identify lysine 159 as a major neddylation site on YAP1. These findings reveal a novel mechanism for neddylation in the regulation of Hippo-YAP signaling., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Hyperuricemia (HUA) is a metabolic disorder characterized by elevated serum uric acid (UA), primarily attributed to the hepatic overproduction and renal underexcretion of UA. Despite the elucidation of molecular pathways associated with this underexcretion, the etiology of HUA remains largely unknown. In our study, using by Uox knockout rats, HUA mouse, and cell line models, we discovered that the increased WWC1 levels were associated with decreased renal UA excretion. Additionally, using knockdown and overexpression approaches, we found that WWC1 inhibited UA excretion in renal tubular epithelial cells. Mechanistically, WWC1 activated the Hippo pathway, leading to phosphorylation and subsequent degradation of the downstream transcription factor YAP1, thereby impairing the ABCG2 and OAT3 expression through transcriptional regulation. Consequently, this reduction led to a decrease in UA excretion in renal tubular epithelial cells. In conclusion, our study has elucidated the role of upregulated WWC1 in renal tubular epithelial cells inhibiting the excretion of UA in the kidneys and causing HUA., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

HIV-1 integration into the human genome is dependent on 3'-processing of the viral DNA. Recently, we reported that the cellular Three Prime Repair Exonuclease 1 (TREX1) enhances HIV-1 integration by degrading the unprocessed viral DNA, while the integration-competent 3'-processed DNA remained resistant. Here, we describe the mechanism by which the 3'-processed HIV-1 DNA resists TREX1-mediated degradation. Our kinetic studies revealed that the rate of cleavage (k cat ) of the 3'-processed DNA was significantly lower (approximately 2-2.5-fold) than the unprocessed HIV-1 DNA by TREX1. The k cat values of human TREX1 for the processed U5 and U3 DNA substrates were 3.8 s -1 and 4.5 s -1 , respectively. In contrast, the unprocessed U5 and U3 substrates were cleaved at 10.2 s -1 and 9.8 s -1 , respectively. The efficiency of degradation (k cat /K m ) of the 3'-processed DNA (U5-70.2 and U3-28.05 pM -1 s -1 ) was also significantly lower than the unprocessed DNA (U5-103.1 and U3-65.3 pM -1 s -1 ). Furthermore, the binding affinity (K d ) of TREX1 was markedly lower (∼2-fold) for the 3'-processed DNA than the unprocessed DNA. Molecular docking and dynamics studies revealed distinct conformational binding modes of TREX1 with the 3'-processed and unprocessed HIV-1 DNA. Particularly, the unprocessed DNA was favorably positioned in the active site with polar interactions with the catalytic residues of TREX1. Additionally, a stable complex was formed between TREX1 and the unprocessed DNA compared the 3'-processed DNA. These results pinpoint the mechanism by which TREX1 preferentially degrades the integration-incompetent HIV-1 DNA and reveal the unique structural and conformational properties of the integration-competent 3'-processed HIV-1 DNA., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest with the content of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The nucleocapsid protein (N) of SARS-CoV-2 is essential for virus replication, genome packaging, evading host immunity, and virus maturation. N is a multidomain protein composed of an independently folded monomeric N-terminal domain that is the primary site for RNA binding and a dimeric C-terminal domain that is essential for efficient phase separation and condensate formation with RNA. The domains are separated by a disordered Ser/Arg-rich region preceding a self-associating Leu-rich helix. Phosphorylation in the Ser/Arg region in infected cells decreases the viscosity of N:RNA condensates promoting viral replication and host immune evasion. The molecular level effect of phosphorylation, however, is missing from our current understanding. Using NMR spectroscopy and analytical ultracentrifugation, we show that phosphorylation destabilizes the self-associating Leu-rich helix 30 amino-acids distant from the phosphorylation site. NMR and gel shift assays demonstrate that RNA binding by the linker is dampened by phosphorylation, whereas RNA binding to the full-length protein is not significantly affected presumably due to retained strong interactions with the primary RNA-binding domain. Introducing a switchable self-associating domain to replace the Leu-rich helix confirms the importance of linker self-association to droplet formation and suggests that phosphorylation not only increases solubility of the positively charged elongated Ser/Arg region as observed in other RNA-binding proteins but can also inhibit self-association of the Leu-rich helix. These data highlight the effect of phosphorylation both at local sites and at a distant self-associating hydrophobic helix in regulating liquid-liquid phase separation of the entire protein., Competing Interests: Conflicts of interests The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Novel components in the noncanonical Hippo pathway that mediate the growth, metastasis, and drug resistance of breast cancer (BC) cells need to be identified. Here, we showed that expression of SAM and SH3 domain-containing protein 1 (SASH1) is negatively correlated with expression of mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) in a subpopulation of patients with luminal-subtype BC. Downregulated SASH1 and upregulated MAP4K4 synergistically regulated the proliferation, migration, and invasion of luminal-subtype BC cells. The expression of LATS2, SASH1, and YAP1 and the phosphorylation of YAP1 were negatively regulated by MAP4K4, and LATS2 then phosphorylated SASH1 to form a novel MAP4K4-LATS2-SASH1-YAP1 cascade. Dephosphorylation of Yes1 associated transcriptional regulator (YAP1), YAP1/TAZ nuclear translocation, and downstream transcriptional regulation of YAP1 were promoted by the combined effects of ectopic MAP4K4 expression and SASH1 silencing. Targeted inhibition of MAP4K4 blocked proliferation, cell migration, and ER signaling both in vitro and in vivo. Our findings reveal a novel MAP4K4-LATS2-SASH1-YAP1 phosphorylation cascade, a noncanonical Hippo pathway that mediates ER signaling, tumorigenesis, and metastasis in breast cancer. Targeted intervention with this noncanonical Hippo pathway may constitute a novel alternative therapeutic approach for endocrine-resistant BC., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest related to the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

We are interested in the contribution of integrins and the extracellular matrix to epithelial differentiation in carcinomas. This study was motivated by our finding that the Hippo effectors YAP and TAZ can sustain the expression of laminin 332 (LM332), the predominant ECM ligand for the integrin β4, in breast carcinoma cells with epithelial differentiation. More specifically, we observed that YAP and TAZ regulate the transcription of the LAMC2 subunit of LM332. Given that the β4-LM332 axis is associated with epithelial differentiation and YAP/TAZ have been implicated in carcinoma de-differentiation, we sought to resolve this paradox. Here, we observed that the β4 integrin sustains the expression of miR-200s that target the transcription factor ZEB1 and that ZEB1 has a pivotal role in determining the nature of YAP/TAZ-mediated transcription. In the presence of β4, ZEB1 expression is repressed enabling YAP/TAZ/TEAD-mediated transcription of LAMC2. The absence of β4, however, induces ZEB1, and ZEB1 binds to the LAMC2 promoter to inhibit LAMC2 transcription. YAP/TAZ-mediated regulation of LAMC2 has important functional consequences because we provide evidence that LM332 enables carcinoma cells to resist ferroptosis in concert with the β4 integrin., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Macrophages play critical roles in inflammation and tissue homeostasis, and their functions are regulated by various autocrine, paracrine, and endocrine factors. We have previously shown that CTRP6, a secreted protein of the C1q family, targets both adipocytes and macrophages to promote obesity-linked inflammation. However, the gene programs and signaling pathways directly regulated by CTRP6 in macrophages remain unknown. Here, we combine transcriptomic and phosphoproteomic analyses to show that CTRP6 activates inflammatory gene programs and signaling pathways in mouse bone marrow-derived macrophages (BMDMs). Treatment of BMDMs with CTRP6 upregulated proinflammatory, and suppressed the antiinflammatory, gene expression. We also showed that CTRP6 activates p44/42-MAPK, p38-MAPK, and NF-κB signaling pathways to promote inflammatory cytokine secretion from BMDMs, and that pharmacologic inhibition of these signaling pathways markedly attenuated the effects of CTRP6. Pretreatment of BMDMs with CTRP6 also sensitized and potentiated the BMDMs response to lipopolysaccharide (LPS)-induced inflammatory signaling and cytokine secretion. Consistent with the metabolic phenotype of proinflammatory macrophages, CTRP6 treatment induced a shift toward aerobic glycolysis and lactate production, reduced oxidative metabolism, and elevated mitochondrial reactive oxygen species production in BMDMs. Importantly, in accordance with our in vitro findings, BMDMs from CTRP6-deficient mice were less inflammatory at baseline and showed a marked suppression of LPS-induced inflammatory gene expression and cytokine secretion. Finally, loss of CTRP6 in mice also dampened LPS-induced inflammation and hypothermia. Collectively, our findings suggest that CTRP6 regulates and primes the macrophage response to inflammatory stimuli and thus may have a role in modulating tissue inflammatory tone in different physiological and disease contexts., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The heterohexameric ATPases associated with diverse cellular activities (AAA)-ATPase Pex1/Pex6 is essential for the formation and maintenance of peroxisomes. Pex1/Pex6, similar to other AAA-ATPases, uses the energy from ATP hydrolysis to mechanically thread substrate proteins through its central pore, thereby unfolding them. In related AAA-ATPase motors, substrates are recruited through binding to the motor's N-terminal domains or N terminally bound cofactors. Here, we use structural and biochemical techniques to characterize the function of the N1 domain in Pex6 from budding yeast, Saccharomyces cerevisiae. We found that although Pex1/ΔN1-Pex6 is an active ATPase in vitro, it does not support Pex1/Pex6 function at the peroxisome in vivo. An X-ray crystal structure of the isolated Pex6 N1 domain shows that the Pex6 N1 domain shares the same fold as the N-terminal domains of PEX1, CDC48, and NSF, despite poor sequence conservation. Integrating this structure with a cryo-EM reconstruction of Pex1/Pex6, AlphaFold2 predictions, and biochemical assays shows that Pex6 N1 mediates binding to both the peroxisomal membrane tether Pex15 and an extended loop from the D2 ATPase domain of Pex1 that influences Pex1/Pex6 heterohexamer stability. Given the direct interactions with both Pex15 and the D2 ATPase domains, the Pex6 N1 domain is poised to coordinate binding of cofactors and substrates with Pex1/Pex6 ATPase activity., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The conserved protein kinase mTOR (mechanistic target of rapamycin) responds to diverse environmental cues to control cell metabolism and promote cell growth, proliferation, and survival as part of two multiprotein complexes, mTOR complex 1 (mTORC1) and mTORC2. Our prior work demonstrated that an alkaline intracellular pH (pHi) increases mTORC2 activity and cell survival in complete media in part by activating AMP-activated protein kinase, a kinase best known to sense energetic stress. It is important to note that an alkaline pHi represents an underappreciated hallmark of cancer cells that promotes their oncogenic behaviors. In addition, mechanisms that control mTORC1 and mTORC2 signaling and function remain incompletely defined, particularly in response to stress conditions. Here, we demonstrate that an alkaline pHi increases phosphatidylinositide 3-kinase (PI3K) activity to promote mTORC1 and mTORC2 signaling in the absence of serum growth factors. Alkaline pHi increases mTORC1 activity through PI3K-Akt signaling, which mediates inhibitory phosphorylation of the upstream proteins tuberous sclerosis complex 2 and proline-rich Akt substrate of 40 kDa and dissociates tuberous sclerosis complex from lysosomal membranes, thus enabling Rheb-mediated activation of mTORC1. Thus, alkaline pHi mimics growth factor-PI3K signaling. Functionally, we also demonstrate that an alkaline pHi increases cap-dependent protein synthesis through inhibitory phosphorylation of eIF4E binding protein 1 and suppresses apoptosis in a PI3K- and mTOR-dependent manner. We speculate that an alkaline pHi promotes a low basal level of cell metabolism (e.g., protein synthesis) that enables cancer cells within growing tumors to proliferate and survive despite limiting growth factors and nutrients, in part through elevated PI3K-mTORC1 and/or PI3K-mTORC2 signaling., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Voltage-gated sodium (Na V ) channels drive the upstroke of the action potential and are comprised of a pore-forming α-subunit and regulatory β-subunits. The β-subunits modulate the gating, trafficking, and pharmacology of the α-subunit. These functions are routinely assessed by ectopic expression in heterologous cells. However, currently available expression systems may not capture the full range of these effects since they contain endogenous β-subunits. To better reveal β-subunit functions, we engineered a human cell line devoid of endogenous Na V β-subunits and their immediate phylogenetic relatives. This new cell line, β-subunit-eliminated eHAP expression (BeHAPe) cells, were derived from haploid eHAP cells by engineering inactivating mutations in the β-subunits SCN1B, SCN2B, SCN3B, and SCN4B, and other subfamily members MPZ (myelin protein zero(P0)), MPZL1, MPZL2, MPZL3, and JAML. In diploid BeHAPe cells, the cardiac Na V α-subunit, Na V 1.5, was highly sensitive to β-subunit modulation and revealed that each β-subunit and even MPZ imparted unique gating properties. Furthermore, combining β1 and β2 with Na V 1.5 generated a sodium channel with hybrid properties, distinct from the effects of the individual subunits. Thus, this approach revealed an expanded ability of β-subunits to regulate Na V 1.5 activity and can be used to improve the characterization of other α/β Na V complexes., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Grim-19 (gene associated with retinoid-IFN-induced mortality 19), the essential component of complex I of mitochondrial respiratory chain, functions as a noncanonical tumor suppressor by controlling apoptosis and energy metabolism. However, additional biological actions of Grim-19 have been recently suggested in male reproduction. We investigated here the expression and functional role of Grim-19 in murine testis. Testicular Grim-19 expression was detected from mouse puberty and increased progressively thereafter, and GRIM-19 protein was observed to be expressed exclusively in interstitial Leydig cells (LCs), with a prominent mitochondrial localization. In vivo lentiviral vector-mediated knockdown of Grim-19 resulted in a significant decrease in testosterone production and triggered aberrant oxidative stress in testis, thus impairing male fertility by inducing germ cell apoptosis and oligozoospermia. The control of testicular steroidogenesis by GRIM-19 was validated using the in vivo knockdown model with isolated primary LCs and in vitro experiments with MA-10 mouse Leydig tumor cells. Mechanistically, we suggest that the negative regulation exerted by GRIM-19 deficiency-induced oxidative stress on steroidogenesis may be the result of two phenomena: a direct effect through inhibition of phosphorylation of steroidogenic acute regulatory protein (StAR) and subsequent impediment to StAR localization in mitochondria and an indirect pathway that is to facilitate the inhibiting role exerted by the extracellular matrix on the steroidogenic capacity of LCs via promotion of integrin activation. Altogether, our observations suggest that Grim-19 plays a potent role in testicular steroidogenesis and that its alterations may contribute to testosterone deficiency-related disorders linked to metabolic stress and male infertility., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Mitochondrial morphology and dynamics maintain mitochondrial integrity by regulating its size, shape, distribution, and connectivity, thereby modulating various cellular processes. Several studies have established a functional link between mitochondrial dynamics, mitophagy, and cell death, but further investigation is needed to identify specific proteins involved in mitochondrial dynamics. Any alteration in the integrity of mitochondria has severe ramifications that include disorders like cancer and neurodegeneration. In this study, we used budding yeast as a model organism and found that Pil1, the major component of the eisosome complex, also localizes to the periphery of mitochondria. Interestingly, the absence of Pil1 causes the branched tubular morphology of mitochondria to be abnormally fused or aggregated, whereas its overexpression leads to mitochondrial fragmentation. Most importantly, pil1Δ cells are defective in mitophagy and bulk autophagy, resulting in elevated levels of reactive oxygen species and protein aggregates. In addition, we show that pil1Δ cells are more prone to cell death. Yeast two-hybrid analysis and co-immunoprecipitations show the interaction of Pil1 with two major proteins in mitochondrial fission, Fis1 and Dnm1. Additionally, our data suggest that the role of Pil1 in maintaining mitochondrial shape is dependent on Fis1 and Dnm1, but it functions independently in mitophagy and cell death pathways. Together, our data suggest that Pil1, an eisosome protein, is a novel regulator of mitochondrial morphology, mitophagy, and cell death., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Respiratory syncytial virus has a negative-sense single-stranded RNA genome constitutively encapsidated by the viral nucleoprotein N, forming a helical nucleocapsid which is the template for viral transcription and replication by the viral polymerase L. Recruitment of L onto the nucleocapsid depends on the viral phosphoprotein P, which is an essential L cofactor. A prerequisite for genome and antigenome encapsidation is the presence of the monomeric, RNA-free, neosynthesized N protein, named N 0 . Stabilization of N 0 depends on the binding of the N-terminal residues of P to its surface, which prevents N oligomerization. However, the mechanism involved in the transition from N 0 -P to nucleocapsid assembly, and thus in the specificity of viral genome encapsidation, is still unknown. Furthermore, the specific role of N oligomerization and RNA in the morphogenesis of viral factories, where viral transcription and replication occur, have not been elucidated although the interaction between P and N complexed to RNA has been shown to be responsible for this process. Here, using a chimeric protein comprising N and the first 40 N-terminal residues of P, we succeeded in purifying a recombinant N 0 -like protein competent for RNA encapsidation in vitro. Our results showed the importance of RNA length for stable encapsidation and revealed that the nature of the 5' end of RNA does not explain the specificity of encapsidation. Finally, we showed that RNA encapsidation is crucial for the in vitro reconstitution of pseudo-viral factories. Together, our findings provide insight into respiratory syncytial virus viral genome encapsidation specificity., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

WW domain-containing E3 Ubiquitin-protein ligase 2 (WWP2) has been found to positively regulate odontoblastic differentiation by monoubiquitinating the transcription factor Kruppel-like factor 5 (KLF5) in a cell culture system. However, the in vivo role of WWP2 in mouse teeth remains unknown. To explore this, here we generated Wwp2 knockout (Wwp2 KO) mice. We found that molars in Wwp2 KO mice exhibited thinner dentin, widened predentin, and reduced numbers of dentinal tubules. In addition, expression of the odontoblast differentiation markers Dspp and Dmp1 was decreased in the odontoblast layers of Wwp2 KO mice. These findings demonstrate that WWP2 may facilitate odontoblast differentiation and dentinogenesis. Furthermore, we show for the first time that phosphatase and tensin homolog (PTEN), a tumor suppressor, is expressed in dental papilla cells and odontoblasts of mouse molars and acts as a negative regulator of odontoblastic differentiation. Further investigation indicated that PTEN is targeted by WWP2 for degradation during odontoblastic differentiation. We demonstrate PTEN physically interacts with and inhibits the transcriptional activity of KLF5 on Dspp and Dmp1. Finally, we found WWP2 was able to suppress the interaction between PTEN and KLF5, which diminished the inhibition effect of PTEN on KLF5. Taken together, this study confirms the essential role of WWP2 and the WWP2-PTEN-KLF5 signaling axis in odontoblast differentiation and dentinogenesis in vivo., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

During spliceosome assembly, the 3' splice site is recognized by sequential U2AF2 complexes, first with Splicing Factor 1 (SF1) and second by the SF3B1 subunit of the U2 small nuclear ribonuclear protein particle. The U2AF2-SF1 interface is well characterized, comprising a U2AF homology motif (UHM) of U2AF2 bound to a U2AF ligand motif (ULM) of SF1. However, the structure of the U2AF2-SF3B1 interface and its importance for pre-mRNA splicing are unknown. To address this knowledge gap, we determined the crystal structure of the U2AF2 UHM bound to a SF3B1 ULM site at 1.8-Å resolution. We discovered a distinctive trajectory of the SF3B1 ULM across the U2AF2 UHM surface, which differs from prior UHM/ULM structures and is expected to modulate the orientations of the full-length proteins. We established that the binding affinity of the U2AF2 UHM for the cocrystallized SF3B1 ULM rivals that of a nearly full-length U2AF2 protein for an N-terminal SF3B1 region. An additional SF3B6 subunit had no detectable effect on the U2AF2-SF3B1 binding affinities. We further showed that key residues at the U2AF2 UHM-SF3B1 ULM interface contribute to coimmunoprecipitation of the splicing factors. Moreover, disrupting the U2AF2-SF3B1 interface changed splicing of representative human transcripts. From analysis of genome-wide data, we found that many of the splice sites coregulated by U2AF2 and SF3B1 differ from those coregulated by U2AF2 and SF1. Taken together, these findings support distinct structural and functional roles for the U2AF2-SF1 and U2AF2-SF3B1 complexes during the pre-mRNA splicing process., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Phosphate homeostasis, mediated by dietary intake, renal absorption, and bone deposition, is incompletely understood because of the uncharacterized roles of numerous implicated protein factors. Here, we identified a novel role for one such element, regulator of G protein signaling 14 (RGS14), suggested by genome-wide association studies to associate with dysregulated Pi levels. We show that human RGS14 possesses a carboxy-terminal PDZ ligand required for sodium phosphate cotransporter 2a (NPT2A) and sodium hydrogen exchanger regulatory factor-1 (NHERF1)-mediated renal Pi transport. In addition, we found using isotope uptake measurements combined with bioluminescence resonance energy transfer assays, siRNA knockdown, pull-down and overlay assays, and molecular modeling that secreted proteins parathyroid hormone (PTH) and fibroblast growth factor 23 inhibited Pi uptake by inducing dissociation of the NPT2A-NHERF1 complex. PTH failed to affect Pi transport in cells expressing RGS14, suggesting that it suppresses hormone-sensitive but not basal Pi uptake. Interestingly, RGS14 did not affect PTH-directed G protein activation or cAMP formation, implying a postreceptor site of action. Further pull-down experiments and direct binding assays indicated that NPT2A and RGS14 bind distinct PDZ domains on NHERF1. We showed that RGS14 expression in human renal proximal tubule epithelial cells blocked the effects of PTH and fibroblast growth factor 23 and stabilized the NPT2A-NHERF1 complex. In contrast, RGS14 genetic variants bearing mutations in the PDZ ligand disrupted RGS14 binding to NHERF1 and subsequent PTH-sensitive Pi transport. In conclusion, these findings identify RGS14 as a novel regulator of hormone-sensitive Pi transport. The results suggest that changes in RGS14 function or abundance may contribute to the hormone resistance and hyperphosphatemia observed in kidney diseases., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The Hippo signaling pathway regulates tissue growth and cell fate, and its dysregulation can induce tumorigenesis. When Hippo is activated by cell-cell contact, extracellular signals, or cell polarity among others, the large tumor suppressor 1 (LATS1) kinase catalyzes inhibitory phosphorylation of the transcriptional coactivator Yes-associated protein (YAP) to maintain YAP in the cytoplasm or promote its degradation. Separately, calmodulin is a Ca 2+ -dependent protein that modulates the activity of target proteins and regulates several signaling cascades; however, its potential role in the Hippo pathway has not been identified. Here, using diverse experimental approaches, including in vitro binding analyses, kinase assays, RT-PCR, and confocal microscopy, we reveal that calmodulin promotes Hippo signaling. We show that purified YAP and LATS1 bind directly to calmodulin and form a Ca 2+ -dependent ternary complex in vitro. Importantly, Ca 2+ /calmodulin directly stimulated the activity of LATS1 kinase. In cultured mammalian cells, we demonstrated that endogenous YAP and LATS1 coimmunoprecipitate with endogenous calmodulin. In cells with activated Hippo signaling, we show that calmodulin antagonism significantly (i) decreases YAP phosphorylation, (ii) increases expression of two Hippo target genes (connective tissue growth factor [CTGF] and cysteine-rich angiogenic inducer 61 [CYR61]) that regulate cell proliferation and tumor progression, and (iii) enhances the interaction of YAP with its major transcription factor, thereby facilitating transcription of target genes. Collectively, our data demonstrate that calmodulin activates the Hippo kinase cascade and inhibits YAP activity via a direct interaction with LATS1 and YAP, thereby uncovering previously unidentified crosstalk between the Ca 2+ /calmodulin and Hippo signaling pathways., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Published by Elsevier Inc.)

The sodium-potassium ATPase (Na/K-ATPase, NKA) establishes ion gradients that facilitate many physiological functions including action potentials and secondary transport processes. NKA comprises a catalytic subunit (alpha) that interacts closely with an essential subunit (beta) and regulatory transmembrane micropeptides called FXYD proteins. In the heart, a key modulatory partner is the FXYD protein phospholemman (PLM, FXYD1), but the stoichiometry of the alpha-beta-PLM regulatory complex is unknown. Here, we used fluorescence lifetime imaging and spectroscopy to investigate the structure, stoichiometry, and affinity of the NKA-regulatory complex. We observed a concentration-dependent binding of the subunits of NKA-PLM regulatory complex, with avid association of the alpha subunit with the essential beta subunit as well as lower affinity alpha-alpha and alpha-PLM interactions. These data provide the first evidence that, in intact live cells, the regulatory complex is composed of two alpha subunits associated with two beta subunits, decorated with two PLM regulatory subunits. Docking and molecular dynamics (MD) simulations generated a structural model of the complex that is consistent with our experimental observations. We propose that alpha-alpha subunit interactions support conformational coupling of the catalytic subunits, which may enhance NKA turnover rate. These observations provide insight into the pathophysiology of heart failure, wherein low NKA expression may be insufficient to support formation of the complete regulatory complex with the stoichiometry (alpha-beta-PLM) 2 ., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Zonula occludens-1 (ZO-1), the major scaffolding protein of tight junctions (TJs), recruits the cytoskeleton-associated proteins cingulin (CGN) and paracingulin (CGNL1) to TJs by binding to their N-terminal ZO-1 interaction motif. The conformation of ZO-1 can be either folded or extended, depending on cytoskeletal tension and intramolecular and intermolecular interactions, and only ZO-1 in the extended conformation recruits the transcription factor DbpA to TJs. However, the sequences of ZO-1 that interact with CGN and CGNL1 and the role of TJ proteins in ZO-1 TJ assembly are not known. Here, we used glutathione-S-transferase pulldowns and immunofluorescence microscopy to show that CGN and CGNL1 bind to the C-terminal ZU5 domain of ZO-1 and that this domain is required for CGN and CGNL1 recruitment to TJs and to phase-separated ZO-1 condensates in cells. We show that KO of CGN, but not CGNL1, results in decreased accumulation of ZO-1 at TJs. Furthermore, ZO-1 lacking the ZU5 domain showed decreased accumulation at TJs, was detectable along lateral contacts, had a higher mobile fraction than full-length ZO-1 by fluorescence recovery after photobleaching analysis, and had a folded conformation, as determined by structured illumination microscopy of its N-terminal and C-terminal ends. The CGN-ZU5 interaction promotes the extended conformation of ZO-1, since binding of the CGN-ZO-1 interaction motif region to ZO-1 resulted in its interaction with DbpA in cells and in vitro. Together, these results show that binding of CGN to the ZU5 domain of ZO-1 promotes ZO-1 stabilization and accumulation at TJs by promoting its extended conformation., Competing Interests: Conflict of interest J. R. T. is a founder and shareholder of Thelium Therapeutics and has served as a consultant for Entrinsic, Immunic, and Kallyope. S. C. has served as a consultant for Immunic. All other authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Mammalian spermatogenesis is a highly coordinated process that requires cooperation between specific proteins to coordinate diverse biological functions. For example, mouse Parkin coregulated gene (PACRG) recruits meiosis-expressed gene 1 (MEIG1) to the manchette during normal spermiogenesis. Here we mutated Y68 of MEIG1 using the CRISPR/cas9 system and examined the biological and physiological consequences in mice. All homozygous mutant males examined were completely infertile, and sperm count was dramatically reduced. The few developed sperm were immotile and displayed multiple abnormalities. Histological staining showed impaired spermiogenesis in these mutant mice. Immunofluorescent staining further revealed that this mutant MEIG1 was still present in the cell body of spermatocytes, but also that more MEIG1 accumulated in the acrosome region of round spermatids. The mutant MEIG1 and a cargo protein of the MEIG1/PACRG complex, sperm-associated antigen 16L (SPAG16L), were no longer found to be present in the manchette; however, localization of the PACRG component was not changed in the mutants. These findings demonstrate that Y68 of MEIG1 is a key amino acid required for PACRG to recruit MEIG1 to the manchette to transport cargo proteins during sperm flagella formation. Given that MEIG1 and PACRG are conserved in humans, small molecules that block MEIG1/PACRG interaction are likely ideal targets for the development of male contraconception drugs., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Nucleic acid-sensing pathways play critical roles in innate immune activation through the production of type I interferon (IFN-I) and proinflammatory cytokines. These factors are required for effective antitumor immune responses. Pharmacological modulators of the pre-mRNA spliceosome splicing factor 3b subunit 1 (SF3B1) are under clinical investigation as cancer cytotoxic agents. However, potential roles of these agents in aberrant RNA generation and subsequent RNA-sensing pathway activation have not been studied. In this study, we observed that SF3B1 pharmacological modulation using pladienolide B (Plad B) induces production of aberrant RNA species and robust IFN-I responses via engagement of the dsRNA sensor retinoic acid-inducible gene I (RIG-I) and downstream interferon regulatory factor 3. We found that Plad B synergized with canonical RIG-I agonism to induce the IFN-I response. In addition, Plad B induced NF-κB responses and secretion of proinflammatory cytokines and chemokines. Finally, we showed that cancer cells bearing the hotspot SF3B1 K700E mutation, which leads to global aberrant splicing, had enhanced IFN-I response to canonical RIG-I agonism. Together, these results demonstrate that pharmacological modulation of SF3B1 in cancer cells can induce an enhanced IFN-I response dependent on RIG-I expression. The study suggests that spliceosome modulation may not only induce direct cancer cell cytotoxicity but also initiate an innate immune response via activation of RNA-sensing pathways., Competing Interests: Conflict of interest All authors were employees of Pfizer, Inc during the duration of this work., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Interferon-γ-inducible factor 16 (IFI16) triggers stimulator of interferon (IFN) genes (STING)-dependent type I IFN production during host antiviral immunity and facilitates p53-dependent apoptosis during suppressing tumorigenesis. We have previously reported that STING-mediated IFI16 degradation negatively regulates type I IFN production. However, it is unknown whether STING also suppresses IFI16/p53-dependent apoptosis via degradation of IFI16. Here, our results from flow cytometry apoptosis detection and immunoblot assays show that IFI16 and nutlin-3, a p53 pathway activator, synergistically induce apoptosis in U2OS and A549 cells. Protein kinase R-triggered phosphorylation of p53 at serine 392 is critical for the IFI16-p53-dependent apoptosis. However, overexpression of STING suppresses p53 serine 392 phosphorylation, p53 transcriptional activity, expression of p53 target genes, and p53-dependent mitochondrial depolarization and apoptosis. In summary, our current study demonstrates that STING-mediated IFI16 degradation negatively regulates IFI16-mediated p53-dependent apoptosis in osteosarcoma and non-small cell lung cancer cells, which suggests a protumorigenic role for STING in certain cancer types because of its potent ability to degrade upstream IFI16., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The Hippo pathway is a key regulatory pathway that is tightly regulated by mechanical cues such as tension, pressure, and contact with the extracellular matrix and other cells. At the distal end of the pathway is the yes-associated protein (YAP), a well-characterized transcriptional regulator. Through binding to transcription factors such as the TEA Domain TFs (TEADs) YAP regulates expression of several genes involved in cell fate, proliferation and death decisions. While the function of YAP as direct transcriptional regulator has been extensively characterized, only a small number of studies examined YAP function as a regulator of gene expression via microRNAs. We utilized bioinformatic approaches, including chromatin immunoprecipitation sequencing and RNA-Seq, to identify potential new targets of YAP regulation and identified miR-30a as a YAP target gene in Schwann cells. We find that YAP binds to the promoter and regulates the expression of miR-30a. Moreover, we identify several YAP-regulated genes that are putative miR-30a targets and focus on two of these, protein tyrosine pohosphatase non-receptor type 13 (PTPN13) and Kruppel like factor 9. We find that YAP regulation of Schwann cell proliferation and death is mediated, to a significant extent, through miR-30a regulation of PTPN13 in Schwann cells. These findings identify a new regulatory function by YAP, mediated by miR-30a, to downregulate expression of PTPN13 and Kruppel like factor 9. These studies expand our understanding of YAP function as a regulator of miRNAs and illustrate the complexity of YAP transcriptional functions., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Endosomal signaling downstream of G-protein-coupled receptors (GPCRs) has emerged as a novel paradigm with important pharmacological and physiological implications. However, our knowledge of the functional consequences of intracellular signaling is incomplete. To begin to address this gap, we combined an optogenetic approach for site-specific generation of the prototypical second messenger generated by active GPCRs, cyclic AMP (cAMP), with unbiased mass-spectrometry-based analysis of the phosphoproteome. We identified 218 unique, high-confidence sites whose phosphorylation is either increased or decreased in response to cAMP elevation. We next determined that the same amount of cAMP produced from the endosomal membrane led to more robust changes in phosphorylation than the plasma membrane. Remarkably, this was true for the entire repertoire of 218 identified targets and irrespective of their annotated subcellular localizations (endosome, cell surface, nucleus, cytosol). Furthermore, we identified a particularly strong endosome bias for a subset of proteins that are dephosphorylated in response to cAMP. Through bioinformatics analysis, we established these targets as putative substrates for protein phosphatase 2A (PP2A), and we propose compartmentalized activation of PP2A by cAMP-responsive kinases as the likely underlying mechanism. Altogether, our study extends the concept that endosomal signaling is a significant functional contributor to cellular responsiveness to cAMP by establishing a unique role for localized cAMP production in defining categorically distinct phosphoresponses., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

More than half a century ago, reversible protein phosphorylation was linked to mitochondrial metabolism through the regulation of pyruvate dehydrogenase. Since this discovery, the number of identified mitochondrial protein phosphorylation sites has increased by orders of magnitude, driven largely by technological advances in mass spectrometry-based phosphoproteomics. However, the majority of these modifications remain uncharacterized, rendering their function and relevance unclear. Nonetheless, recent studies have shown that disruption of resident mitochondrial protein phosphatases causes substantial metabolic dysfunction across organisms, suggesting that proper management of mitochondrial phosphorylation is vital for organellar and organismal homeostasis. While these data suggest that phosphorylation within mitochondria is of critical importance, significant gaps remain in our knowledge of how these modifications influence organellar function. Here, we curate publicly available datasets to map the extent of protein phosphorylation within mammalian mitochondria and to highlight the known functions of mitochondrial-resident phosphatases. We further propose models by which phosphorylation may affect mitochondrial enzyme activities, protein import and processing, and overall organellar homeostasis., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The type II sodium-dependent phosphate cotransporter (NPT2A) mediates renal phosphate uptake. The NPT2A is regulated by parathyroid hormone (PTH) and fibroblast growth factor 23, which requires Na + /H + exchange regulatory factor-1 (NHERF1), a multidomain PDZ-containing phosphoprotein. Phosphocycling controls the association between NHERF1 and the NPT2A. Here, we characterize the critical involvement of G protein-coupled receptor kinase 6A (GRK6A) in mediating PTH-sensitive phosphate transport by targeted phosphorylation coupled with NHERF1 conformational rearrangement, which in turn allows phosphorylation at a secondary site. GRK6A, through its carboxy-terminal PDZ recognition motif, binds NHERF1 PDZ1 with greater affinity than PDZ2. However, the association between NHERF1 PDZ2 and GRK6A is necessary for PTH action. Ser 162 , a PKCα phosphorylation site in PDZ2, regulates the binding affinity between PDZ2 and GRK6A. Substitution of Ser 162 with alanine (S 162 A) blocks the PTH action but does not disrupt the interaction between NHERF1 and the NPT2A. Replacement of Ser 162 with aspartic acid (S 162 D) abrogates the interaction between NHERF1 and the NPT2A and concurrently PTH action. We used amber codon suppression to generate a phosphorylated Ser 162 (pSer 162 )-PDZ2 variant. K D values determined by fluorescence anisotropy indicate that incorporation of pSer 162 increased the binding affinity to the carboxy terminus of GRK6A 2-fold compared with WT PDZ2. Molecular dynamics simulations predict formation of an electrostatic network between pSer 162 and Asp 183 of PDZ2 and Arg at position -1 of the GRK6A PDZ-binding motif. Our results suggest that PDZ2 plays a regulatory role in PTH-sensitive NPT2A-mediated phosphate transport and phosphorylation of Ser 162 in PDZ2 modulates the interaction with GRK6A., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Published by Elsevier Inc.)

While details remain unclear, initiation of woven bone mineralization is believed to be mediated by collagen and potentially nucleated by bone sialoprotein (BSP). Interestingly, our recent publication showed that BSP and type XI collagen form complexes in mineralizing osteoblastic cultures. To learn more, we examined the protein composition of extracellular sites of de novo hydroxyapatite deposition which were enriched in BSP and Col11a1 containing an alternatively spliced "6b" exonal sequence. An alternate splice variant "6a" sequence was not similarly co-localized. BSP and Col11a1 co-purify upon ion-exchange chromatography or immunoprecipitation. Binding of the Col11a1 "6b" exonal sequence to bone sialoprotein was demonstrated with overlapping peptides. Peptide 3, containing three unique lysine-triplet sequences, displayed the greatest binding to osteoblastic cultures; peptides containing fewer lysine triplet motifs or derived from the "6a" exon yielded dramatically lower binding. Similar results were obtained with 6-carboxyfluorescein (FAM)-conjugated peptides and western blots containing extracts from osteoblastic cultures. Mass spectroscopic mapping demonstrated that FAM-peptide 3 bound to 90 kDa BSP and its 18 to 60 kDa fragments, as well as to 110 kDa nucleolin. In osteoblastic cultures, FAM-peptide 3 localized to biomineralization foci (site of BSP) and to nucleoli (site of nucleolin). In bone sections, biotin-labeled peptide 3 bound to sites of new bone formation which were co-labeled with anti-BSP antibodies. These results establish the fluorescent peptide 3 conjugate as the first nonantibody-based method to identify BSP on western blots and in/on cells. Further examination of the "6b" splice variant interactions will likely reveal new insights into bone mineralization during development., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The C1q and TNF related 4 (C1QTNF4) protein is a structurally unique member of the C1QTNF family, a family of secreted proteins that have structural homology with both complement C1q and the tumor necrosis factor superfamily. C1QTNF4 has been linked to the autoimmune disease systemic lupus erythematosus through genetic studies; however, its role in immunity and inflammation remains poorly defined and a cell surface receptor of C1QTNF4 has yet to be identified. Here we report identification of nucleolin as a cell surface receptor of C1QTNF4 using mass spectrometric analysis. Additionally, we present evidence that the interaction between C1QTNF4 and nucleolin is mediated by the second C1q-like domain of C1QTNF4 and the C terminus of nucleolin. We show that monocytes and B cells are target cells of C1QTNF4 and observe extensive binding to dead cells. Imaging flow cytometry experiments in monocytes show that C1QTNF4 becomes actively internalized upon cell binding. Our results suggest that nucleolin may serve as a docking molecule for C1QTNF4 and act in a context-dependent manner through coreceptors. Taken together, these findings further our understanding of C1QTNF4's function in the healthy immune system and how dysfunction may contribute to the development of systemic lupus erythematosus., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Programmed cell death protein 1 (PD-1) is a critical inhibitory receptor that limits excessive T cell responses. Cancer cells have evolved to evade these immunoregulatory mechanisms by upregulating PD-1 ligands and preventing T cell-mediated anti-tumor responses. Consequently, therapeutic blockade of PD-1 enhances T cell-mediated anti-tumor immunity, but many patients do not respond and a significant proportion develop inflammatory toxicities. To improve anti-cancer therapy, it is critical to reveal the mechanisms by which PD-1 regulates T cell responses. We performed global quantitative phosphoproteomic interrogation of PD-1 signaling in T cells. By complementing our analysis with functional validation assays, we show that PD-1 targets tyrosine phosphosites that mediate proximal T cell receptor signaling, cytoskeletal organization, and immune synapse formation. PD-1 ligation also led to differential phosphorylation of serine and threonine sites within proteins regulating T cell activation, gene expression, and protein translation. In silico predictions revealed that kinase/substrate relationships engaged downstream of PD-1 ligation. These insights uncover the phosphoproteomic landscape of PD-1-triggered pathways and reveal novel PD-1 substrates that modulate diverse T cell functions and may serve as future therapeutic targets. These data are a useful resource in the design of future PD-1-targeting therapeutic approaches., Competing Interests: Conflict of interest—A. M. received research support from NTB Pharma. The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Tocheva et al.)

Histone recognition by "reader" modules serves as a fundamental mechanism in epigenetic regulation. Previous studies have shown that Spindlin1 is a reader of histone H3K4me3 as well as "K4me3-R8me2a" and promotes transcription of rDNA or Wnt/TCF4 target genes. Here we show that Spindlin1 also acts as a potent reader of histone H3 "K4me3-K9me3/2" bivalent methylation pattern. Calorimetric titration revealed a binding affinity of 16 nm between Spindlin1 and H3 "K4me3-K9me3" peptide, which is one to three orders of magnitude stronger than most other histone readout events at peptide level. Structural studies revealed concurrent recognition of H3K4me3 and H3K9me3/2 by aromatic pockets 2 and 1 of Spindlin1, respectively. Epigenomic profiling studies showed that Spindlin1 colocalizes with both H3K4me3 and H3K9me3 peaks in a subset of genes enriched in biological processes of transcription and its regulation. Moreover, the distribution of Spindlin1 peaks is primarily associated with H3K4me3 but not H3K9me3, which suggests that Spindlin1 is a downstream effector of H3K4me3 generated in heterochromatic regions. Collectively, our work calls attention to an intriguing function of Spindlin1 as a potent H3 "K4me3-K9me3/2" bivalent mark reader, thereby balancing gene expression and silencing in H3K9me3/2-enriched regions., Competing Interests: Conflict of interest—The authors declare that they have no conflict of interest.

Primary cilia are generated through the extension of the microtubule-based axoneme. Centrosomal protein 104 (CEP104) localizes to the tip of the elongating axoneme, and CEP104 mutations are linked to a ciliopathy, Joubert syndrome. Thus, CEP104 has been implicated in ciliogenesis. However, the mechanism by which CEP104 regulates ciliogenesis remains elusive. We report here that CEP104 is critical for cilium elongation but not for initiating ciliogenesis. We also demonstrated that the tumor-overexpressed gene (TOG) domain of CEP104 exhibits microtubule-polymerizing activity and that this activity is essential for the cilium-elongating activity of CEP104. Knockdown/rescue experiments showed that the N-terminal jelly-roll (JR) fold partially contributes to cilium-elongating activity of CEP104, but neither the zinc-finger region nor the S X IP motif is required for this activity. CEP104 binds to a centriole-capping protein, CP110, through the zinc-finger region and to a microtubule plus-end-binding protein, EB1, through the S X IP motif, indicating that the binding of CP110 and EB1 is dispensable for the cilium-elongating activity of CEP104. Moreover, CEP104 depletion does not affect CP110 removal from the mother centriole, which suggests that CEP104 functions after the removal of CP110. Last, we also showed that CEP104 is required for the ciliary entry of Smoothened and export of GPR161 upon Hedgehog signal activation and that the TOG domain plays a critical role in this activity. Our results define the roles of the individual domains of CEP104 in its functions in cilium elongation and Hedgehog signaling and should enhance our understanding of the mechanism underlying CEP104 mutation-associated ciliopathies., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Yamazoe et al.)

The multifaceted adaptor protein β-arr1 (β-arrestin1) promotes activation of focal adhesion kinase (FAK) by the chemokine receptor CXCR4, facilitating chemotaxis. This function of β-arr1 requires the assistance of the adaptor protein STAM1 (signal-transducing adaptor molecule 1) because disruption of the interaction between STAM1 and β-arr1 reduces CXCR4-mediated activation of FAK and chemotaxis. To begin to understand the mechanism by which β-arr1 together with STAM1 activates FAK, we used site-directed spin-labeling EPR spectroscopy-based studies coupled with bioluminescence resonance energy transfer-based cellular studies to show that STAM1 is recruited to activated β-arr1 by binding to a novel surface on β-arr1 at the base of the finger loop, at a site that is distinct from the receptor-binding site. Expression of a STAM1-deficient binding β-arr1 mutant that is still able to bind to CXCR4 significantly reduced CXCL12-induced activation of FAK but had no impact on ERK-1/2 activation. We provide evidence of a novel surface at the base of the finger loop that dictates non-GPCR interactions specifying β-arrestin-dependent signaling by a GPCR. This surface might represent a previously unidentified switch region that engages with effector molecules to drive β-arrestin signaling., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Zhuo et al.)

Leukocyte recruitment is a universal feature of tissue inflammation and regulated by the interactions of chemokines with their G protein-coupled receptors. Activation of CC chemokine receptor 2 (CCR2) by its cognate chemokine ligands, including CC chemokine ligand 2 (CCL2), plays a central role in recruitment of monocytes in several inflammatory diseases. In this study, we used phosphoproteomics to conduct an unbiased characterization of the signaling network resulting from CCL2 activation of CCR2. Using data-independent acquisition MS analysis, we quantified both the proteome and phosphoproteome in FlpIn-HEK293T cells stably expressing CCR2 at six time points after activation with CCL2. Differential expression analysis identified 699 significantly regulated phosphorylation sites on 441 proteins. As expected, many of these proteins are known to participate in canonical signal transduction pathways and in the regulation of actin cytoskeleton dynamics, including numerous guanine nucleotide exchange factors and GTPase-activating proteins. Moreover, we identified regulated phosphorylation sites in numerous proteins that function in the nucleus, including several constituents of the nuclear pore complex. The results of this study provide an unprecedented level of detail of CCR2 signaling and identify potential targets for regulation of CCR2 function., (© 2020 Huang et al.)

Crystallization of recombinant proteins has been fundamental to our understanding of protein function, dysfunction, and molecular recognition. However, this information has often been gleaned under extremely nonphysiological protein, salt, and H + concentrations. Here, we describe the development of a robust Inka1-Box (iBox)-PAK4cat system that spontaneously crystallizes in several mammalian cell types. The semi-quantitative assay described here allows the measurement of in vivo protein-protein interactions using a novel GFP-linked reporter system that produces fluorescent readouts from protein crystals. We combined this assay with in vitro X-ray crystallography and molecular dynamics studies to characterize the molecular determinants of the interaction between the PDZ2 domain of Na + /H + exchange regulatory cofactor NHE-RF1 (NHERF1) and cystic fibrosis transmembrane conductance regulator (CFTR), a protein complex pertinent to the genetic disease cystic fibrosis. These experiments revealed the crystal structure of the extended PDZ domain of NHERF1 and indicated, contrary to what has been previously reported, that residue selection at positions -1 and -3 of the PDZ-binding motif influences the affinity and specificity of the NHERF1 PDZ2-CFTR interaction. Our results suggest that this system could be utilized to screen additional protein-protein interactions, provided they can be accommodated within the spacious iBox-PAK4cat lattice., (© 2020 Martin et al.)

Animal cells use pattern-recognition receptors (PRRs) to detect specific pathogens. Pathogen detection mounts an appropriate immune response, including interferon and cytokine induction. The intracellular PRR-signaling pathways that detect DNA viruses have been characterized, particularly in myeloid cells. In these pathways, cGMP-AMP synthase (cGAS) and the pyrin and HIN domain family member (PYHIN) protein interferon-γ-inducible protein 16 (IFI16) detect DNA and signal via stimulator of interferon genes protein (STING). However, although airway epithelial cells are frontline sentinels in detecting pathogens, information on how they respond to DNA viruses is limited, and the roles of PYHIN proteins in these cells are unknown. Here, we examined expression and activities of cGAS, STING, and PYHINs in human lung epithelial cells. A549 epithelial cells, commonly used for RNA-sensing studies, failed to respond to DNA because they lacked STING expression, and ectopic STING expression restored a cGAS-dependent DNA response in these cells. In contrast, NuLi-1 immortalized human bronchial epithelial cells did express STING, which was activated after DNA stimulation and mediated DNA-dependent gene induction. PYHIN1, which like IFI16 has been proposed to be a viral DNA sensor, was the only PYHIN protein expressed in both airway epithelial cell types. However, rather than having a role in DNA sensing, PYHIN1 induced proinflammatory cytokines in response to interleukin-1 (IL-1) or tumor necrosis factor α (TNFα) stimulation. Of note, PYHIN1, via its HIN domain, directly induced IL-6 and TNFα transcription, revealing that PYHIN proteins play a role in proinflammatory gene induction in airway epithelial cells., (© 2020 Massa et al.)

Embryonic large molecule derived from yolk sac (ELYS) is a constituent protein of nuclear pores. It initiates assembly of nuclear pore complexes into functional nuclear pores toward the end of mitosis. Using cellular, molecular, and genetic tools, including fluorescence and Electron microscopy, quantitative PCR, and RNAi-mediated depletion, we report here that the ELYS ortholog ( d Elys) plays critical roles during Drosophila development. d Elys localized to the nuclear rim in interphase cells, but during mitosis it was absent from kinetochores and enveloped chromatin. We observed that RNAi-mediated dElys depletion leads to aberrant development and, at the cellular level, to defects in the nuclear pore and nuclear lamina assembly. Further genetic analyses indicated that dElys depletion re-activates the Dorsal (NF-κB) pathway during late larval stages. Re-activated Dorsal caused untimely expression of the Dorsal target genes in the post-embryonic stages. We also demonstrate that activated Dorsal triggers apoptosis during later developmental stages by up-regulating the pro-apoptotic genes reaper and hid The apoptosis induced by Reaper and Hid was probably the underlying cause for developmental abnormalities observed upon dElys depletion. Moreover, we noted that d Elys has conserved structural features, but contains a noncanonical AT-hook-like motif through which it strongly binds to DNA. Together, our results uncover a novel epistatic interaction that regulates Dorsal dynamics by d Elys during development., (© 2020 Mehta et al.)

The Na + /H + exchange regulatory cofactor 1 (NHERF1) protein modulates the assembly and intracellular trafficking of several transmembrane G protein-coupled receptors (GPCRs) and ion transport proteins with the membrane-cytoskeleton adapter protein ezrin. Here, we applied solution NMR and small-angle neutron scattering (SANS) to structurally characterize full-length NHERF1 and disease-associated variants that are implicated in impaired phosphate homeostasis. Using NMR, we mapped the modular architecture of NHERF1, which is composed of two structurally-independent PDZ domains that are connected by a flexible, disordered linker. We observed that the ultra-long and disordered C-terminal tail of NHERF1 has a type 1 PDZ-binding motif that interacts weakly with the proximal, second PDZ domain to form a dynamically autoinhibited structure. Using ensemble-optimized analysis of SANS data, we extracted the molecular size distribution of structures from the extensive conformational space sampled by the flexible chain. Our results revealed that NHERF1 is a diffuse ensemble of variable PDZ domain configurations and a disordered C-terminal tail. The joint NMR/SANS data analyses of three disease variants (L110V, R153Q, and E225K) revealed significant differences in the local PDZ domain structures and in the global conformations compared with the WT protein. Furthermore, we show that the substitutions affect the affinity and kinetics of NHERF1 binding to ezrin and to a C-terminal peptide from G protein-coupled receptor kinase 6A (GRK6A). These findings provide important insight into the modulation of the intrinsic flexibility of NHERF1 by disease-associated point mutations that alter the dynamic assembly of signaling complexes., (© 2019 by The American Society for Biochemistry and Molecular Biology, Inc.)

Neurite outgrowth requires coordinated cytoskeletal rearrangements in the growth cone and directional membrane delivery from the neuronal soma. As an essential Rho guanine nucleotide exchange factor (GEF), TRIO is necessary for cytoskeletal dynamics during neurite outgrowth, but its participation in the membrane delivery is unclear. Using co-localization studies, live-cell imaging, and fluorescence recovery after photobleaching analysis, along with neurite outgrowth assay and various biochemical approaches, we here report that in mouse cerebellar granule neurons, TRIO protein pools at the Golgi and regulates membrane trafficking by controlling the directional maintenance of both RAB8 (member RAS oncogene family 8)- and RAB10-positive membrane vesicles. We found that the spectrin repeats in Golgi-resident TRIO confer RAB8 and RAB10 activation by interacting with and activating the RAB GEF RABIN8. Constitutively active RAB8 or RAB10 could partially restore the neurite outgrowth of TRIO-deficient cerebellar granule neurons, suggesting that TRIO-regulated membrane trafficking has an important functional role in neurite outgrowth. Our results also suggest cross-talk between Rho GEF and Rab GEF in controlling both cytoskeletal dynamics and membrane trafficking during neuronal development. They further highlight how protein pools localized to specific organelles regulate crucial cellular activities and functions. In conclusion, our findings indicate that TRIO regulates membrane trafficking during neurite outgrowth in coordination with its GEF-dependent function in controlling cytoskeletal dynamics via Rho GTPases., (© 2019 Tao et al.)

Dynamic regulation of the actin cytoskeleton is an essential feature of cell motility. Action of Enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP), a family of conserved actin-elongating proteins, is an important aspect of regulation of the actin cytoskeletal architecture at the leading edge that controls membrane protrusion and cell motility. In this study, we performed mutagenesis experiments in overexpression and knockdown-rescue settings to provide, for the first time, direct evidence of the role of the actin-binding protein profilin1 (Pfn1) in VASP-mediated regulation of cell motility. We found that VASP's interaction with Pfn1 is promoted by cell-substrate adhesion and requires down-regulation of PKA activity. Our experimental data further suggest that PKA-mediated Ser 137 phosphorylation of Pfn1 potentially negatively regulates the Pfn1-VASP interaction. Finally, Pfn1's ability to be phosphorylated on Ser 137 was partly responsible for the anti-migratory action elicited by exposing cells to a cAMP/PKA agonist. On the basis of these findings, we propose a mechanism of adhesion-protrusion coupling in cell motility that involves dynamic regulation of Pfn1 by PKA activity., (© 2019 Gau et al.)

Focal adhesions (FAs) are specialized sites where intracellular cytoskeleton elements connect to the extracellular matrix and thereby control cell motility. FA assembly depends on various scaffold proteins, including the G protein-coupled receptor kinase-interacting protein 1 (GIT1), paxillin, and liprin-α. Although liprin-α and paxillin are known to competitively interact with GIT1, the molecular basis governing these interactions remains elusive. To uncover the underlying mechanisms of how GIT1 is involved in FA assembly by alternatively binding to liprin-α and paxillin, here we solved the crystal structures of GIT1 in complex with liprin-α and paxillin at 1.8 and 2.6 Å resolutions, respectively. These structures revealed that the paxillin-binding domain (PBD) of GIT1 employs distinct binding modes to recognize a single α-helix of liprin-α and the LD4 motif of paxillin. Structure-based design of protein variants produced two binding-deficient GIT1 variants; specifically, these variants lost the ability to interact with liprin-α only or with both liprin-α and paxillin. Expressing the GIT1 variants in COS7 cells, we discovered that the two PBD-meditated interactions play different roles in either recruiting GIT1 to FA or facilitating FA assembly. Additionally, we demonstrate that, unlike for the known binding mode of the FAT domain to LD motifs, the PBD of GIT1 uses different surface patches to achieve high selectivity in LD motif recognition. In summary, our results have uncovered the mechanisms by which GIT1's PBD recognizes cognate paxillin and liprin-α structures, information we anticipate will be useful for future investigations of GIT1-protein interactions in cells., (© 2019 Liang et al.)

Diabetes promotes the posttranslational modification of proteins by O- linked addition of GlcNAc ( O- GlcNAcylation) to Ser/Thr residues of proteins and thereby contributes to diabetic complications. In the retina of diabetic mice, the repressor of mRNA translation, eIF4E-binding protein 1 (4E-BP1), is O- GlcNAcylated, and sequestration of the cap-binding protein eukaryotic translation initiation factor (eIF4E) is enhanced. O- GlcNAcylation has also been detected on several eukaryotic translation initiation factors and ribosomal proteins. However, the functional consequence of this modification is unknown. Here, using ribosome profiling, we evaluated the effect of enhanced O- GlcNAcylation on retinal gene expression. Mice receiving thiamet G (TMG), an inhibitor of the O- GlcNAc hydrolase O- GlcNAcase, exhibited enhanced retinal protein O- GlcNAcylation. The principal effect of TMG on retinal gene expression was observed in ribosome-associated mRNAs ( i.e. mRNAs undergoing translation), as less than 1% of mRNAs exhibited changes in abundance. Remarkably, ∼19% of the transcriptome exhibited TMG-induced changes in ribosome occupancy, with 1912 mRNAs having reduced and 1683 mRNAs having increased translational rates. In the retina, the effect of O- GlcNAcase inhibition on translation of specific mitochondrial proteins, including superoxide dismutase 2 (SOD2), depended on 4E-BP1/2. O- GlcNAcylation enhanced cellular respiration and promoted mitochondrial superoxide levels in WT cells, and 4E-BP1/2 deletion prevented O- GlcNAcylation-induced mitochondrial superoxide in cells in culture and in the retina. The retina of diabetic WT mice exhibited increased reactive oxygen species levels, an effect not observed in diabetic 4E-BP1/2-deficient mice. These findings provide evidence for a mechanism whereby diabetes-induced O- GlcNAcylation promotes oxidative stress in the retina by altering the selection of mRNAs for translation., (© 2019 Dierschke et al.)

Na + -H + exchanger regulatory factor-1 (NHERF1) is a PDZ protein that scaffolds membrane proteins, including sodium-phosphate co-transport protein 2A (NPT2A) at the plasma membrane. NHERF1 is a phosphoprotein with 40 Ser and Thr residues. Here, using tandem MS analysis, we characterized the sites of parathyroid hormone (PTH)-induced NHERF1 phosphorylation and identified 10 high-confidence phosphorylation sites. Ala replacement at Ser 46 , Ser 162 , Ser 181 , Ser 269 , Ser 280 , Ser 291 , Thr 293 , Ser 299 , and Ser 302 did not affect phosphate uptake, but S290A substitution abolished PTH-dependent phosphate transport. Unexpectedly, Ser 290 was rapidly dephosphorylated and rephosphorylated after PTH stimulation, and we found that protein phosphatase 1α (PP1α), which binds NHERF1 through a conserved VxF/W PP1 motif, dephosphorylates Ser 290 Mutating 257 VPF 259 eliminated PP1 binding and blunted dephosphorylation. Tautomycetin blocked PP1 activity and abrogated PTH-sensitive phosphate transport. Using fluorescence lifetime imaging (FLIM), we observed that PTH paradoxically and transiently elevates intracellular phosphate. Added phosphate blocked PP1α-mediated Ser 290 dephosphorylation of recombinant NHERF1. Hydrogen-deuterium exchange MS revealed that β-sheets in NHERF1's PDZ2 domain display lower deuterium uptake than those in the structurally similar PDZ1, implying that PDZ1 is more cloistered. Dephosphorylated NHERF1 exhibited faster exchange at C-terminal residues suggesting that NHERF1 dephosphorylation precedes Ser 290 rephosphorylation. Our results show that PP1α and NHERF1 form a holoenzyme and that a multiprotein kinase cascade involving G protein-coupled receptor kinase 6A controls the Ser 290 phosphorylation status of NHERF1 and regulates PTH-sensitive, NPT2A-mediated phosphate uptake. These findings reveal how reversible phosphorylation modifies protein conformation and function and the biochemical mechanisms underlying PTH control of phosphate transport., (© 2019 Zhang et al.)

Cardiovascular disease (CVD) remains the leading cause of death globally, and heart failure is a major component of CVD-related morbidity and mortality. The development of cardiac hypertrophy in response to hemodynamic overload is initially considered to be beneficial; however, this adaptive response is limited and, in the presence of prolonged stress, will transition to heart failure. Yes-associated protein (YAP), the central downstream effector of the Hippo signaling pathway, regulates proliferation and survival in mammalian cells. Our previous work demonstrated that cardiac-specific loss of YAP leads to increased cardiomyocyte (CM) apoptosis and impaired CM hypertrophy during chronic myocardial infarction (MI) in the mouse heart. Because of its documented cardioprotective effects, we sought to determine the importance of YAP in response to acute pressure overload (PO). Our results indicate that endogenous YAP is activated in the heart during acute PO. YAP activation that depended upon RhoA was also observed in CMs subjected to cyclic stretch. To examine the function of endogenous YAP during acute PO, Yap +/ flox ;Cre α- MHC (YAP-CHKO) and Yap +/ flox mice were subjected to transverse aortic constriction (TAC). We found that YAP-CHKO mice had attenuated cardiac hypertrophy and significant increases in CM apoptosis and fibrosis that correlated with worsened cardiac function after 1 week of TAC. Loss of CM YAP also impaired activation of the cardioprotective kinase Akt, which may underlie the YAP-CHKO phenotype. Together, these data indicate a prohypertrophic, prosurvival function of endogenous YAP and suggest a critical role for CM YAP in the adaptive response to acute PO., (© 2019 Byun et al.)

Bone morphogenetic protein (BMP) signaling is critical in renal development and disease. In animal models of chronic kidney disease (CKD), re-activation of BMP signaling is reported to be protective by promoting renal repair and regeneration. Clinical use of recombinant BMPs, however, requires harmful doses to achieve efficacy and is costly because of BMPs' complex synthesis. Therefore, alternative strategies are needed to harness the beneficial effects of BMP signaling in CKD. Key aspects of the BMP signaling pathway can be regulated by both extracellular and intracellular molecules. In particular, secreted proteins like noggin and chordin inhibit BMP activity, whereas kielin/chordin-like proteins (KCP) enhance it and attenuate kidney fibrosis or CKD. Clinical development of KCP, however, is precluded by its size and complexity. Therefore, we propose an alternative strategy to enhance BMP signaling by using small molecules, which are simpler to synthesize and more cost-effective. To address our objective, here we developed a small-molecule high-throughput screen (HTS) with human renal cells having an integrated luciferase construct highly responsive to BMPs. We demonstrate the activity of a potent benzoxazole compound, sb4, that rapidly stimulated BMP signaling in these cells. Activation of BMP signaling by sb4 increased the phosphorylation of key second messengers (SMAD-1/5/9) and also increased expression of direct target genes (inhibitors of DNA binding, Id1 and Id3 ) in canonical BMP signaling. Our results underscore the feasibility of utilizing HTS to identify compounds that mimic key downstream events of BMP signaling in renal cells and have yielded a lead BMP agonist., (© 2019 Bradford et al.)

The transcription elongation and pre-mRNA splicing factor Tat-SF1 associates with the U2 small nuclear ribonucleoprotein (snRNP) of the spliceosome. However, the direct binding partner and underlying interactions mediating the Tat-SF1-U2 snRNP association remain unknown. Here, we identified SF3b1 as a Tat-SF1-interacting subunit of the U2 snRNP. Our 1.1 Å resolution crystal structure revealed that Tat-SF1 contains a U2AF homology motif (UHM) protein-protein interaction module. We demonstrated that Tat-SF1 preferentially and directly binds the SF3b1 subunit compared with other U2AF ligand motif (ULM)-containing splicing factors, and further established that SF3b1 association depends on the integrity of the Tat-SF1 UHM. We next compared the Tat-SF1-binding affinities for each of the five known SF3b1 ULMs and then determined the structures of representative high- and low-affinity SF3b1 ULM complexes with the Tat-SF1 UHM at 1.9 Å and 2.1 Å resolutions, respectively. These structures revealed a canonical UHM-ULM interface, comprising a Tat-SF1 binding pocket for a ULM tryptophan (SF3b1 Trp 338 ) and electrostatic interactions with a basic ULM tail. Importantly, we found that SF3b1 regulates Tat-SF1 levels and that these two factors influence expression of overlapping representative transcripts, consistent with a functional partnership of Tat-SF1 and SF3b1. Altogether, these results define a new molecular interface of the Tat-SF1-U2 snRNP complex for gene regulation., (© 2019 Loerch et al.)

The mammalian lipin 1 phosphatidate phosphatase is a key regulatory enzyme in lipid metabolism. By catalyzing phosphatidate dephosphorylation, which produces diacylglycerol, the enzyme plays a major role in the synthesis of triacylglycerol and membrane phospholipids. The importance of lipin 1 to lipid metabolism is exemplified by cellular defects and lipid-based diseases associated with its loss or overexpression. Phosphorylation of lipin 1 governs whether it is associated with the cytoplasm apart from its substrate or with the endoplasmic reticulum membrane where its enzyme reaction occurs. Lipin 1β is phosphorylated on multiple sites, but less than 10% of them are ascribed to a specific protein kinase. Here, we demonstrate that lipin 1β is a bona fide substrate for casein kinase II (CKII), a protein kinase that is essential to viability and cell cycle progression. Phosphoamino acid analysis and phosphopeptide mapping revealed that lipin 1β is phosphorylated by CKII on multiple serine and threonine residues, with the former being major sites. Mutational analysis of lipin 1β and its peptides indicated that Ser-285 and Ser-287 are both phosphorylated by CKII. Substitutions of Ser-285 and Ser-287 with nonphosphorylatable alanine attenuated the interaction of lipin 1β with 14-3-3β protein, a regulatory hub that facilitates the cytoplasmic localization of phosphorylated lipin 1. These findings advance our understanding of how phosphorylation of lipin 1β phosphatidate phosphatase regulates its interaction with 14-3-3β protein and intracellular localization and uncover a mechanism by which CKII regulates cellular physiology., (© 2019 Hennessy et al.)