Pathogenic Yersinia contain a virulence plasmid that encodes genes for intracellular effectors, which neutralize the host immune response. One effector, YopM, is necessary for Yersinia virulence, but its function in host cells is unknown. To identify potential cellular pathways affected by YopM, proteins that co-immunoprecipitate with YopM in mammalian cells were isolated and identified by mass spectrometry. Results demonstrate that two kinases, protein kinase C-like 2 (PRK2) and ribosomal S6 protein kinase 1 (RSK1), interact directly with YopM. These two kinases associate only when YopM is present, and expression of YopM in cells stimulates the activity of both kinases. RSK1 is activated directly by interaction with YopM, and RSK1 kinase activity is required for YopM-stimulated PRK2 activity. YopM activation of RSK1 occurs independently of the actions of YopJ on the MAPK pathway. YopM is also required for Yersinia-induced changes in RSK1 mobility in infected macrophage cells. These results identify the first intracellular targets of YopM and suggest YopM acts to stimulate the activity of PRK2 and RSK1.

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The myotubularin (MTM) family constitutes one of the most highly conserved protein-tyrosine phosphatase subfamilies in eukaryotes. MTM1, the archetypal member of this family, is mutated in X-linked myotubular myopathy, whereas mutations in the MTMrelated (MTMR)2 gene cause the type 4B1 Charcot-Marie-Tooth disease, a severe hereditary motor and sensory neuropathy. In this study, we identified a protein that specifically interacts with MTMR2 but not MTM1. The interacting protein was shown by mass spectrometry to be MTMR5, a catalytically inactive member of the MTM family. We also demonstrate that MTMR2 interacts with MTMR5 via its coiled-coil domain and that mutations in the coiledcoil domain of either MTMR2 or MTMR5 abrogate this interaction. Through this interaction, MTMR5 increases the enzymatic activity of MTMR2 and dictates its subcellular localization. This article demonstrates an active MTM member being regulated by an inactive family member.T he myotubularin (MTM) family constitutes one of the largest and most highly conserved protein-tyrosine phosphatase (PTP) subfamilies in eukaryotes (1-3). The human MTM family of phosphatases includes MTM1͞MTM-related (MTMR)1͞ MTMR2, MTMR3͞MTMR4, and MTMR6͞MTMR7͞MTMR8 subgroups (1, 3). The consensus CX 5 R active site motif is found in the MTM family and the sequence "CSDGWDR" is invariant within all of the enzymatically active members of this family. Most PTPs use phosphoproteins as substrates and specifically dephosphorylate substrates containing only phosphotyrosine sites. Other phosphatases, collectively known as dual-specificity phosphatases, are capable of removing phosphoserines͞ threonines and phosphotyrosines from protein substrates.Initially, MTM1 was reported to be a dual-specificity phosphatase (4-6). However, we and others have demonstrated that MTM1 utilizes the lipid second messenger, phosphatidylinositol 3-phosphate [PI(3)P], as a physiological substrate (7,8). Recent findings demonstrate that other MTMR phosphatases MTMR1, MTMR2, MTMR3, MTMR4, and MTMR6 also dephosphorylate PI(3)P, suggesting that activity toward this substrate is common to all active MTM family members (9-12). MTMR2 and MTMR3 have also been shown to dephosphorylate phosphatidylinositol 3,5-bisphosphate (9, 13). PI(3)P plays a key role in membrane trafficking͞vesicular transport processes and serves as a targeting mechanism for proteins containing specific PI(3)P-binding modules such as Fab1͞YOTB͞Vac1p͞EEA1 (FYVE), pleckstrin homology (PH), and Phox homology domains (14-17).To date, two MTMR proteins have been associated with human diseases. The MTM1 gene on chromosome Xq28 is mutated in X-linked myotubular myopathy, a severe congenital muscular disorder characterized by hypotonia and generalized muscle weakness in newborn males (18,19 MTM1 and MTMR2 are highly similar proteins (64% identity, 76% similarity), use the same physiologic substrate, and have a ubiquitous expression pattern (6, 9-12). However, mutations in MTM1 and MTMR2 cause different diseases with different target tissues...

Tuberous sclerosis complex (TSC) is a genetic disease caused by mutations in either TSC1 or TSC2 tumor suppressor genes. TSC1 and TSC2 (also known as hamartin and tuberin, respectively) form a functional complex and negatively regulate cell growth by inhibiting protein synthesis. 14-3-3 binds to TSC2 and may inhibit TSC2 function. We Tuberous sclerosis complex (TSC)1 is an autosomal dominant genetic disorder occurring in 1/6,000 individuals. Studies of TSC patients demonstrate that mutation of either TSC1 or TSC2 is responsible for TSC. Mutation of TSC1 and TSC2 each accounts for ϳ50% of TSC cases (1). TSC is characterized by the development of benign hamartomas in many organs, including brain, kidney, heart, skin, and eyes. Although malignancy rarely develops in TSC hamartomas, brain hamartomas produce the most serious clinical complication and often result in mental retardation, seizures, and autism. Other symptoms include renal dysfunction, dermatological abnormalities, and heart problems (2).Studies of TSC patients and animal models support the hypothesis that TSC1 and TSC2 are tumor suppressor genes. Loss of function of either the TSC1 or TSC2 gene product is the underlying molecular basis for the pathogenesis of TSC (3, 4). Eker rats contain a heterozygous mutation in TSC2 and have very high incidence of tumors, especially renal carcinomas (5). Tumors in Eker rats are generated by mutation of the wild type allele of the TSC2 gene. Homozygous deletion of either TSC1 or TSC2 in mice produces an embryonic lethal phenotype, demonstrating an essential function in development (6, 7). Heterozygous deletion of either TSC1 or TSC2 shows 100% incidence of renal carcinomas and a significant increase of carcinomas in other tissues.TSC1 and TSC2 gene products are also known as hamartin and tuberin, respectively. Mutation in TSC1 or TSC2 results in similar phenotypes, suggesting that the two proteins function in the same pathway (8). In fact, TSC1 and TSC2 form a physical complex, and the TSC1⅐TSC2 complex is functionally important in vivo. Many disease-derived mutations in TSC2 weaken the complex formation with TSC1 (9 -12). However, the cellular functions of TSC1 and TSC2 were not known until recent genetic studies in Drosophila demonstrated that TSC1 and TSC2 play a major negative role in the regulation of cell growth (13-15). Mutations of either TSC1 or TSC2 significantly increase cell size in Drosophila. Genetic epistatic studies indicate that TSC1⅐TSC2 acts downstream from the insulin receptor, which plays a major role in cell growth control and cell size regulation. Phosphatidylinositol 3-kinase (PI 3-kinase) is a major downstream effector of the insulin receptor. Activation of the PI 3-kinase-Akt pathway plays an important role in cell proliferation, oncogenic transformation, cell survival, and cell size control (16).We have reported recently that TSC2 is a direct physiological target of Akt. Akt phosphorylates TSC2 on multiple sites and inactivates TSC2 function (12). We identified that serine 939, serine 10...

hYVH1 [human orthologue of YVH1 (yeast VH1-related phosphatase)] is an atypical dual-specificity phosphatase that is widely conserved throughout evolution. Deletion studies in yeast have suggested a role for this phosphatase in regulating cell growth. However, the role of the human orthologue is unknown. The present study used MS to identify Hsp70 (heat-shock protein 70) as a novel hYVH1-binding partner. The interaction was confirmed using endogenous co-immunoprecipitation experiments and direct binding of purified proteins. Endogenous Hsp70 and hYVH1 proteins were also found to co-localize specifically to the perinuclear region in response to heat stress. Domain deletion studies revealed that the ATPase effector domain of Hsp70 and the zinc-binding domain of hYVH1 are required for the interaction, indicating that this association is not simply a chaperone-substrate complex. Thermal phosphatase assays revealed hYVH1 activity to be unaffected by heat and only marginally affected by non-reducing conditions, in contrast with the archetypical dual-specificity phosphatase VHR (VH1-related protein). In addition, Hsp70 is capable of increasing the phosphatase activity of hYVH1 towards an exogenous substrate under non-reducing conditions. Furthermore, the expression of hYVH1 repressed cell death induced by heat shock, H2O2 and Fas receptor activation but not cisplatin. Co-expression of hYVH1 with Hsp70 further enhanced cell survival. Meanwhile, expression of a catalytically inactive hYVH1 or a hYVH1 variant that is unable to interact with Hsp70 failed to protect cells from the various stress conditions. The results suggest that hYVH1 is a novel cell survival phosphatase that co-operates with Hsp70 to positively affect cell viability in response to cellular insults.

The presence of late embryogenesis abundant (LEA) proteins in plants and animals has been linked to their ability to tolerate a variety of environmental stresses. Among animals, encysted embryos of the brine shrimp Artemia franciscana are among the most stress resistant eukaryotes, and for that reason it is considered to be an extremophile. The study presented here demonstrates that these embryos contain multiple group 1 LEA proteins with masses of 21, 19, 15.5 and 13 kDa. The LEA proteins first appear in diapause-destined embryos, beginning at ∼4 days post-fertilization, but not in nauplii-destined embryos. After resumption of embryonic development, the LEA proteins decline slowly in the desiccation resistant encysted stages, then disappear rapidly as the embryo emerges from its shell. LEA proteins are absent in fully emerged embryos, larvae and adults. They are abundant in mitochondria of encysted embryos, but barely detectable in nuclei and absent from yolk platelets. LEA proteins were also detected in dormant embryos of six other species of Artemia from hypersaline environments around the world. This study enhances our knowledge of the group 1 LEA proteins in stress tolerant crustacean embryos.

Src homology 3 domain (SH3)-containing proline-rich protein kinase (SPRK)/mixed-lineage kinase (MLK)-3 is a serine/threonine kinase that upon overexpression in mammalian cells activates the c-Jun NH 2 -terminal kinase pathway. The mechanisms by which SPRK activity is regulated are not well understood. The small Rho family GTPases, Rac and Cdc42, have been shown to bind and modulate the activities of signaling proteins, including SPRK, which contain Cdc42/Rac interactive binding motifs. Coexpression of SPRK and activated Cdc42 increases SPRKs activity. SPRKs Cdc42/Rac interactive binding-like motif contains six of the eight consensus residues. Using a site-directed mutagenesis approach, we show that SPRK contains a functional Cdc42/Rac interactive binding motif that is required for SPRKs association with and activation by Cdc42. However, experiments using a SPRK variant that lacks the COOH-terminal zipper region/basic stretch suggest that this region may also contribute to Cdc42 binding. Unlike the PAK family of protein kinases, we find that the activation of SPRK by Cdc42 cannot be recapitulated in an in vitro system using purified, recombinant proteins. Comparative phosphopeptide mapping demonstrates that coexpression of activated Cdc42 with SPRK alters the in vivo serine/threonine phosphorylation pattern of SPRK suggesting that the mechanism by which Cdc42 increases SPRKs catalytic activity involves a change in the in vivo phosphorylation of SPRK. This is, to the best of our knowledge, the first demonstrated example of a Cdc42-mediated change in the in vivo phosphorylation of a protein kinase. These studies suggest an additional component or cellular environment is required for SPRK activation by Cdc42.

HSF1 (heat-shock factor 1) plays an essential role in mediating the appropriate cellular response to diverse forms of physiological stresses. However, it is not clear how HSF1 is regulated by interacting proteins under normal and stressful conditions. In the present study, Hsc70 (heat-shock cognate 70) was identified as a HSF1-interacting protein using the TAP (tandem affinity purification) system and MS. HSF1 can interact with Hsc70 in vivo and directly in vitro. Interestingly, Hsc70 is required for the regulation of HSF1 during heat stress and subsequent target gene expression in mammalian cells. Moreover, cells transfected with siRNAs (small interfering RNAs) targeted to Hsc70 showed greatly decreased HSF1 activation with expression of HSF1 target genes being dramatically reduced. Finally, loss of Hsc70 expression in cells resulted in an increase in stress-induced apoptosis. These results indicate that Hsc70 is a necessary and critical regulator of HSF1 activities.