Endogenous inhibitor proteins that connect Ser/Thr kinases and phosphatases in cell signaling.

Protein phosphatase activity acts as a primary determinant of the extent and duration of phosphorylation of cellular proteins in response to physiological stimuli. Ser/Thr protein phosphatase-1 (PP1) belongs to the PPP superfamily, and is associated with regulatory subunits that confer substrate specificity, allosteric regulation, and subcellular compartmentalization. In addition, all eukaryotic cells contain multiple heat-stable proteins that originally were thought to inhibit phosphatase catalytic subunits released from the regulatory subunits, as a fail-safe mechanism. However, discovery of C-kinase-activated PP1 inhibitor, Mr of 17 kDa (CPI-17) required fresh thinking about the endogenous inhibitors as specific regulators of particular phosphatase complexes, acting in addition to, not instead of, regulatory subunits. The cellular actions of the endogenous inhibitors are controlled by phosphorylation, connecting them to kinase pathways. More recent progress has unveiled additional functions of PP1 inhibitor-2 (I-2), including regulation of protein kinases. Transcriptional mechanisms govern the expression levels of CPI-17 in response to stimuli. If true for other inhibitor proteins, they have the potential of being diagnostic markers for pathological conditions. We discuss specific examples of PP1 inhibitor proteins regulating particular cellular functions and the rationale for incorporating phosphatase inhibitor proteins in development of new therapeutic strategies

Synergistic inhibition of human melanoma proliferation by combination treatment with B-Raf inhibitor BAY43-9006 and mTOR inhibitor Rapamycin

BACKGROUND: Targeted inhibition of protein kinases is now acknowledged as an effective approach for cancer therapy. However, targeted therapies probably have limited success because cancer cells have alternate pathways for survival and proliferation thereby avoiding inhibition. We tested the hypothesis that combination of targeted agents would be more effective than single agents in arresting melanoma cell proliferation. METHODS: We evaluated whether BAY43-9006, an inhibitor of the B-Raf kinase, and rapamycin, an inhibitor of the mTOR kinase, would inhibit serum-stimulated proliferation of human melanoma cell lines, either alone or in combination. Proliferation was measured by quantitating melanoma cell numbers with a luciferase for ATP. Phosphorylation of proteins downstream of targeted kinase(s) was assayed by immunoblots. Statistical significance was determined with the Student-T test. Isobologram analysis was performed to distinguish additive versus synergistic effects of combinations of drugs. RESULTS: Serum-stimulated proliferation of multiple human melanoma cell lines was inhibited by BAY43-9006 and by rapamycin. Melanoma cells containing the B-Raf mutation V599E were more sensitive than cells with wild-type B-raf to 10 nM doses of both BAY43-9006 and rapamycin. Regardless of B-Raf mutational status, the combination of low dose rapamycin and BAY43-9006 synergistically inhibited melanoma cell proliferation. As expected, rapamycin inhibited the phosphorylation of mTOR substrates, p70S6K and 4EBP1, and BAY43-9006 inhibited phosphorylation of ERK, which is dependent on B-Raf activity. We also observed unexpected rapamycin inhibition of the phosphorylation of ERK, as well as BAY43-9006 inhibition of the phosphorylation of mTOR substrates, p70S6K and 4EBP1. CONCLUSION: There was synergistic inhibition of melanoma cell proliferation by the combination of rapamycin and BAY 43-9006, and unexpected inhibition of two signaling pathways by agents thought to target only one of those pathways. These results indicate that combinations of inhibitors of mTOR and of the B-raf signaling pathways may be more effective as a treatment for melanoma than use of either agent alone

Regulatory crosstalk by protein kinases on CFTR trafficking and activity

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a member of the ATP binding cassette (ABC) transporter superfamily that functions as a cAMP-activated chloride ion channel in fluid-transporting epithelia. There is abundant evidence that CFTR activity (i.e., channel opening and closing) is regulated by protein kinases and phosphatases via phosphorylation and dephosphorylation. Here, we review recent evidence for the role of protein kinases in regulation of CFTR delivery to and retention in the plasma membrane. We review this information in a broader context of regulation of other transporters by protein kinases because the overall functional output of transporters involves the integrated control of both their number at the plasma membrane and their specific activity. While many details of the regulation of intracellular distribution of CFTR and other transporters remain to be elucidated, we hope that this review will motivate research providing new insights into how protein kinases control membrane transport to impact health and disease.Work supported by the Portuguese Fundação para a Ciência e Tecnologia (fellowship grant SFRH/BSAB/105741/2014—to CF, grant PTDC/SAU-ORG/119782/2010—to PJ, and centre grant UID/MULTI/04046/2013—to BioISI); Gilead Génese PGG/039/2014 and ERS Romain Pauwels 2012 (to CF); CFF SWIATE14P0 (to AS); NIH NIDDK P30 072506 Basic and Translational Studies of Cystic Fibrosis (P&F to AS)

Association of the tensin N-terminal protein-tyrosine phosphatase domain with the alpha isoform of protein phosphatase-1 in focal adhesions

Focal adhesions attach cultured cells to the extracellular matrix, and we found endogenous protein phosphatase-1alpha isoform (PP1alpha) localized in adhesions across the entire area of adherent fibroblasts. However, in fibroblasts migrating into a scrape wound or spreading after replating PP1alpha did not appear in adhesions near the leading edge but was recruited into other adhesions coincident in time and space with incorporation of tensin. Endogenous tensin and PP1alpha co-precipitated from cell lysates with isoform-specific PP1 antibodies. Chemical cross-linking of focal adhesion preparations with Lomant\u27s reagent demonstrated molecular proximity of endogenous PP1alpha and tensin, whereas neither focal adhesion kinase nor vinculin was cross-linked and co-precipitated with PP1alpha, suggesting distinct spatial subdomains within adhesions. Transient expression of truncated tensin showed the N-terminal 360 residues, which comprise a protein-tyrosine phosphatase domain, alone were sufficient for isoform-selective co-precipitation of co-expressed PP1alpha. Human prostate cancer PC3 cells are deficient in tensin relative to fibroblasts and have fewer, mostly peripheral adhesions. Transient expression of green fluorescent protein tensin in these cancer cells induced formation of adhesions and recruited endogenous PP1alpha into those adhesions. Thus, the protein-tyrosine phosphatase domain of tensin exhibits isoform-specific association with PP1alpha in a restricted spatial region of adhesions that are formed during cell migration

Human Protein Phosphatase PP6 Regulatory Subunits Provide Sit4-Dependent and Rapamycin–Sensitive Sap Function in Saccharomyces cerevisiae

In the budding yeast Saccharomyces cerevisiae the protein phosphatase Sit4 and four associated proteins (Sap4, Sap155, Sap185, and Sap190) mediate G1 to S cell cycle progression and a number of signaling events controlled by the target of rapamycin TOR signaling cascade. Sit4 and the Sap proteins are ubiquitously conserved and their human orthologs, PP6 and three PP6R proteins, share significant sequence identity with their yeast counterparts. However, relatively little is known about the functions of the PP6 and PP6R proteins in mammalian cells. Here we demonstrate that the human PP6R proteins physically interact with Sit4 when expressed in yeast cells. Remarkably, expression of PP6R2 and PP6R3 but not expression of PP6R1 rescues the growth defect and rapamycin hypersensitivity of yeast cells lacking all four Saps, and these effects require Sit4. Moreover, PP6R2 and PP6R3 enhance cyclin G1 gene expression and DNA synthesis, and partially abrogate the G1 cell cycle delay and the budding defect of the yeast quadruple sap mutant strain. In contrast, the human PP6R proteins only modestly support nitrogen catabolite gene expression and are unable to restore normal levels of eIF2α phosphorylation in the quadruple sap mutant strain. These results illustrate that the human PP6-associated proteins are capable of providing distinct rapamycin-sensitive and Sit4-dependent Sap functions in the heterologous context of the yeast cell. We hypothesize that the human Saps may play analogous roles in mTORC1-PP6 signaling events in metazoans

A conserved domain for glycogen binding in protein phosphatase-1 targeting subunits

AbstractThe skeletal muscle glycogen-binding subunit (GM) of protein phosphatase-1 (PP1) is the founding member of a family of proteins that tether the PP1 catalytic subunit (PP1C) to glycogen and promote the dephosphorylation of glycogen synthase. A hydrophobic sequence (called here the VFV motif) is conserved among GM, the liver subunit GL, and the widely expressed subunits, PTG, R5 and U5. This study analyzed the role of this VFV motif in binding to glycogen and PP1C. Glutathione S-transferase (GST) fusions with the N-terminal domain of GM (GST-GM(1–240)) and with the full length R5 protein (GST-R5) both bound to glycogen in a co-sedimentation assay. In contrast, GST itself did not bind to glycogen. A single residue substitution in GST-GM(1–240), F155A, reduced glycogen binding by 40%. Double residue substitutions V150A/F155A and F155A/V159A resulted in greater reductions (60–70%) in glycogen binding, showing these hydrophobic residues influenced the protein-glycogen interaction. The wild type and V150A/F155A fusion proteins were digested by trypsin into the same sized fragments at the same rate. Furthermore, the wild type and mutated GST-GM proteins as well as GST-R5 bound equivalent amounts of PP1C, in either pull-down or far-Western assays. These results demonstrated retention of overall tertiary structure by the mutated fusion proteins, and indicated that glycogen and PP1C binding are independent of one another. A 68 residue segment of R5 encompassing the VFV motif was sufficient to produce glycogen binding when fused to GST. This motif, that is in bacterial and fungal starch metabolizing enzymes, probably has been conserved during evolution as a functional domain for binding glycogen and starch

Discovery and characterization of small molecules that target the Ral GTPase

The Ras-like GTPases RalA and B are important drivers of tumor growth and metastasis. Chemicals that block Ral function would be valuable as research tools and for cancer therapeutics. Here, we used protein structure analysis and virtual screening to identify drug-like molecules that bind a site on the GDP-form of Ral. Compounds RBC6, RBC8 and RBC10 inhibited Ral binding to its effector RalBP1, Ral-mediated cell spreading in murine fibroblasts and anchorage-independent growth of human cancer cell lines. Binding of RBC8 derivative BQU57 to RalB was confirmed by isothermal titration calorimetry, surface plasma resonance and 15N-HSQC NMR. RBC8 and BQU57 show selectivity for Ral relative to Ras or Rho and inhibit xenograft tumor growth similar to depletion of Ral by siRNA. Our results show the utility of structure-based discovery for development of therapeutics for Ral-dependent cancers

Identification and characterization of two distinct PPP1R2 isoforms in human spermatozoa

Background: Protein Ser/Thr Phosphatase PPP1CC2 is an alternatively spliced isoform of PPP1C that is highly enriched in testis and selectively expressed in sperm. Addition of the phosphatase inhibitor toxins okadaic acid or calyculin A to caput and caudal sperm triggers and stimulates motility, respectively. Thus, the endogenous mechanisms of phosphatase inhibition are fundamental for controlling sperm function and should be characterized. Preliminary results have shown a protein phosphatase inhibitor activity resembling PPP1R2 in bovine and primate spermatozoa. Results: Here we show conclusively, for the first time, that PPP1R2 is present in sperm. In addition, we have also identified a novel protein, PPP1R2P3. The latter was previously thought to be an intron-less pseudogene. We show that the protein corresponding to the pseudogene is expressed. It has PPP1 inhibitory potency similar to PPP1R2. The potential phosphosites in PPP1R2 are substituted by non-phosphorylable residues, T73P and S87R, in PPP1R2P3. We also confirm that PPP1R2/PPP1R2P3 are phosphorylated at Ser121 and Ser122, and report a novel phosphorylation site, Ser127. Subfractionation of sperm structures show that PPP1CC2, PPP1R2/PPP1R2P3 are located in the head and tail structures. Conclusions: The conclusive identification and localization of sperm PPP1R2 and PPP1R2P3 lays the basis for future studies on their roles in acrosome reaction, sperm motility and hyperactivation. An intriguing possibility is that a switch in PPP1CC2 inhibitory subunits could be the trigger for sperm motility in the epididymis and/or sperm hyperactivation in the female reproductive tract.publishe