Matthew J. Wishart

A single mutation converts a novel phosphotyrosine binding domain into a dual-specificity phosphatase

Dual-specificity protein-tyrosine phosphatases (dsPTPases) have been implicated in the inactivation of mitogen-activated protein kinases (MAPKs). We have identified a novel phosphoserine/threonine/tyrosine-binding protein (STYX) that is related in amino acid sequence to dsPTPases, except for the substitution of Gly for Cys in the conserved dsPTPase catalytic loop (HCXXGXXR(S/T)). cDNA subcloning and Northern blot analysis in mouse shows poly(A) hybridization bands of 4.6, 2.4, 1.5, and 1.2 kilobases, with highest abundance in skeletal muscle, testis, and heart. Polymerase chain reaction amplification of reverse-transcribed poly(A) RNA revealed an alternatively spliced form of STYX containing a unique carboxyl terminus. Bacterially expressed STYX is incapable of hydrolyzing Tyr(P)-containing substrates; however, mutation of Gly to Cys (G120C), which structurally mimics the active site of dsPTPases, confers phosphatase activity to this molecule. STYX-G120C mutant hydrolyzes p-nitrophenyl phosphate and dephosphorylates both Tyr(P) and Thr(P) residues of peptide sequences of MAPK homologues. The kinetic parameters of dephosphorylation are similar to human dsPTPase, Vaccinia H1-related, including inhibition by vanadate. We believe this is the first example of a naturally occurring “dominant negative” phosphotyrosine/serine/threonine-binding protein which is structurally related to dsPTPases.

PTEN and myotubularin phosphatases: from 3-phosphoinositide dephosphorylation to disease

The phosphatase and tensin homolog deleted on chromosome ten (PTEN) and myotubularin (MTM1) represent subfamilies of protein tyrosine phosphatases whose principal physiological substrates are D3-phosphorylated inositol phospholipids. As lipid phosphatases, PTEN- and MTM1-related (MTMR) proteins dephosphorylate the products of phosphoinositide 3-kinases and antagonize downstream effectors that utilize 3-phosphoinositides as ligands for protein targeting domains or allosteric activation. Here, we describe the cellular mechanisms of PTEN and MTMR function and their role in the etiology of cancer and other human diseases.

A Family of Putative Tumor Suppressors Is Structurally and Functionally Conserved in Humans and Yeast

In Saccharomyces cerevisiae the CDC14 gene is essential for cell cycle progression. Strains carrying thecdc14-1 ts allele enter the cell cycle and arrest at restrictive temperatures. We have identified two human cDNAs encoding proteins which share sequence identity to the yeast CDC14p. The cell cycle arrest in cdc14-1 ts can be specifically complemented by the human cDNAs suggesting that they are functionally equivalent to the yeast CDC14 gene. Another clone identified in the search for human CDC14-like proteins corresponded to the putative tumor suppressor gene PTEN/MMAC1 (phosphatase and tensin homologue deleted on chromosome 10 or mutated in multiple advanced cancers 1). Analysis of the PTEN/MMAC1 showed that it did not complement the cdc14-1 ts allele and that it is more closely related to the yeast open reading frame YNL128W. Human CDC14p and PTEN/MMAC1 were expressed as recombinant proteins, and both were shown to have kinetic properties characteristic of dual specific phosphatases. The human CDC14p was localized in the nucleus while PTEN/MMAC1 has been reported to be localized in the cytoplasm. Our results suggest that CDC14 and YNL128W/PTEN/MMAC1 represent two related, but distinct, families of human and yeast phosphatases.

Phoxy Lipids: Revealing PX Domains as Phosphoinositide Binding Modules

and functional insights into the PX domain as a novel The coordination of cellular signaling events requires phosphoinositide binding module. both spatial and temporal regulation. A recurring theme PX Domains as Regulators of Membrane among eukaryotic organisms is the use of phosphoinosiand Protein Trafficking tide-specific binding domains to direct proteins to disThe mechanisms underlying membrane trafficking and crete sites within cells where their function(s) are revesicular fusion have been intensively studied using the quired. To date, a number of distinct, highly conserved vacuolar sorting pathway of the budding yeast, Saccharphosphoinositide binding motifs have been identified, omyces cerevisiae, as a model system. Vam7p, a the most well-characterized of which include the C2 PX-containing protein that is an essential component of (PKC conserved region 2), ENTH (epsin N-terminal hovacuolar sorting, is related to the SNAP-23/25 mammamology) (Ford et al., 2001; Itoh et al., 2001), FYVE (Fab1, lian t-SNAREs (Schultz et al., 2000). SNARE proteins, YOTB, Vac1, and EEA1), and PH (pleckstrin homology) which are found on both cytoplasmic donor vesicles domains (Schultz et al., 2000; see references in reviews (v-SNAREs) and the acceptor organelles to which they by Gillooly et al. 2001; Hurley and Meyer, 2001; and are targeted (t-SNAREs), serve to modulate the specificOdorizzi et al., 2000). These domains recruit proteins to ity of docking and fusion between donor and acceptor specific regions in cells via their interactions with inositol membranes. Vam7p has an N-terminal PX domain, a lipids and may also serve as allosteric regulators of C-terminal coiled-coil t-SNARE motif (Schultz et al., enzyme activity or protein-protein interactions. 2000), and localizes to the vacuolar membrane as part The PX domain was initially identified as a conserved of a vacuolar SNARE complex (Sato et al., 1998; Ungermotif of 130 residues within the p40 and p47 mann and Wickner, 1998). Vam7p has previously been subunits of the neutrophil NADPH oxidase superoxide shown to physically interact with Vam3p, which also generating complex (Ponting, 1996). The PX motif can localizes to vacuolar membranes (Sato et al., 1998; Unalso be found in a wide variety of proteins involved in germann and Wickner, 1998). Mutation of conserved cell signaling pathways (phospholipases D1 and D2, PI residues within the Vam7p PX domain create synthetic 3-kinase, and Spo14p), vesicular trafficking and yeast vacuolar sorting defects when combined with a tempervacuolar morphology (human sorting nexins; yeast ature-sensitive vam3 allele, suggesting that the PX doVps5p, Vps17p, Vam7p, and Mvp1p), and control of main is necessary for Vam7p function (Sato et al., 1998). yeast bud emergence and cell polarity (Bem1p and Further, Vam7p does not contain known membrane tarBem3p) (Ponting, 1996). Currently, at least 57 human geting motifs, prompting the question of how it associand 15 yeast proteins that contain PX domains have ates with vacuolar membranes. In an effort to identify been identified (Schultz et al., 2000). the role of the PX domain of Vam7p in vacuolar sorting, Although the function of PX domains has remained Cheever and colleagues have investigated whether it unclear, previous studies have implied a possible role may serve as a targeting module to direct the localization in regulating the subcellular localization of their correof Vam7p (Cheever et al., 2001). sponding proteins. For example, a significant number In this report, several lines of evidence corroborate of PX proteins are localized to membranes or vesicular that the PX domain functions to localize Vam7p to vacustructures within cells (Schultz et al., 2000). In the case olar membranes (Cheever et al., 2001). First, either a PX of the NADPH oxidase complex, translocation from the domain point mutation or deletion results in mislocalizacytosol to membranes occurs upon activation of the tion of Vam7p to the cytoplasm. Because a Vam7p muneutrophil respiratory burst response (see references tant lacking the C-terminal coiled-coil motif required for in Babior, 1999). In addition, many of the PX proteins its interaction with Vam3p still localizes to the vacuolar involved in membrane trafficking processes do not conmembrane, the authors conclude that the PX domain tain recognizable membrane targeting motifs (Schultz alone is both necessary and sufficient to direct memet al., 2000). Taken together, these observations have brane targeting of Vam7p. It is of note that a Vam7p provided a strong rationale for investigating whether PX mutant localizes to endosomes in a yeast strain in which domains might function as membrane targeting modendosome-to-vacuole sorting is blocked (vps4 ). Here, ules. In a series of papers in Nature Cell Biology this they further observe that this localization is similar to month, the PX domains of the NADPH oxidase compothat of FYVE-domain-containing proteins, which are nents, p40 and p47, the yeast vacuolar SNARE, known to target the membrane phosphoinositide, PI(3)P

Gathering STYX: phosphatase-like form predicts functions for unique protein-interaction domains

The effects of tyrosine phosphorylation are manifested and regulated through protein domains that bind to specific phosphotyrosine motifs. STYX is a unique modular domain found within proteins implicated in mediating the effects of tyrosine phosphorylation in vivo. Individual STYX domains are not catalytically active; however, they resemble protein tyrosine phosphatase (PTP) domains and, like PTPs, contain core sequences that recognize phosphorylated substrates. Thus, the STYX domain adds to the repertoire of modular domains that can mediate intracellular signaling in response to protein phosphorylation.

PTEN and myotubularin phosphoinositide phosphatases: bringing bioinformatics to the lab bench

Phosphoinositides play an integral role in a diverse array of cellular signaling processes. Although considerable effort has been directed toward characterizing the kinases that produce inositol lipid second messengers, the study of phosphatases that oppose these kinases remains limited. Current research is focused on the identification of novel lipid phosphatases such as PTEN and myotubularin, their physiologic substrates, signaling pathways and links to human diseases. The use of bioinformatics in conjunction with genetic analyses in model organisms will be essential in elucidating the roles of these enzymes in regulating phosphoinositide-mediated cellular signaling.

The archetype STYX/dead-phosphatase complexes with a spermatid mRNA-binding protein and is essential for normal sperm production

Differentiation of spermatids into spermatozoa is regulated via phosphorylated RNA-binding proteins that modulate the expression of stage-specific mRNAs. We demonstrate that the phosphoserine, -threonine or -tyrosine, interaction protein, Styx, complexes with a testicular RNA-binding protein and is essential for normal spermiogenesis. Ablation of Styx expression in mouse disrupts round and elongating spermatid development, resulting in a >1,000-fold decrease in spermatozoa production. Moreover, Styx−/− males are infertile because of structural head abnormalities in residual epididymal sperm. Immunoprecipitation of Styx with Crhsp-24, a phosphorylated RNA-binding protein implicated in translational repression of histone mRNAs, provides a strategy for regulating posttranscriptional gene expression.

Purification and identification of a 28-kDa calcium-regulated heat-stable protein. A novel secretagogue-regulated phosphoprotein in exocrine pancreas.

This study reports the purification and identification of a novel 28 kDa phosphoprotein from rat pancreatic acini, previously described as being highly regulated by calcium mobilizing secretagogues, which we have designated calcium-regulated heat-stable protein 28 (CRHSP-28). Internal amino acid sequences of purified CRHSP-28 were obtained following trypsin digestion and found to match with >95% identity the predicted amino acid sequence of a novel cDNA recently identified as being highly expressed in human breast carcinomas. Verification that this cDNA codes for human CRHSP-28 was demonstrated by the ability of antiserum raised against purified rat CRHSP-28 to recognize the recombinant human protein when expressed in bacteria. Furthermore, this antibody was found to specifically react with CRHSP-28 in rat acini following one- and two-dimensional electrophoresis and underwent a marked acidic shift in mobility after cholecystokinin stimulation, a phenomenon indicative of an increase in its phosphorylation. CRHSP-28 is predicted to be extremely hydrophilic, is phosphorylated entirely on serine residues, and bears little homology to any known proteins. Finally, the distribution of the CRHSP-28 protein in various rat tissues revealed that although it was present at low levels in almost all tissues, it was most highly expressed in pancreas, followed by the gastric, intestinal, and colonic mucosa. In view of its relative abundance throughout the digestive system and its apparent regulation by calcium-mobilizing agents, this protein may provide valuable insight into the mechanism(s) of calcium signaling in these tissues.

Identification of calcineurin regulated phosphorylation sites on CRHSP-24

CRHSP-24 is a prominently regulated phosphoprotein in pancreatic acinar cells where it is the major substrate for the serine/threonine protein phosphatase, calcineurin, in response to secretagogues. We now identify the four regulated sites of CRHSP-24 phosphorylation as serines 30, 32, 41, and 52 and show that Ser(30) and Ser(32) are directly dephosphorylated by calcineurin. Coordinate phosphorylation/dephosphorylation of these four serines explains the multiple phosphorylated isoforms of CRHSP-24 present in acinar cells and provides a molecular framework to study CRHSP-24 regulation by secretagogues and growth factor-induced kinases and phosphatases in vivo.