The intracellular domain of human protein tyrosine phosphatase beta (HPTP beta) (44 kDa) was expressed in bacteria, purified using epitope 'tagging' immunoaffinity chromatography, and characterized with respect to kinetic profile, substrate specificity and potential modulators of enzyme activity. A chromogenic assay based on the Malachite Green method was employed for the detection of inorganic phosphate (Pi) released from phosphopeptides by HPTP beta. This assay, modified so as to improve its sensitivity, was adapted to a 96-well microtitre plate format, and provided linear detection between 50 and 1000 pmol of Pi. The cytoplasmic domain of HPTP beta was strongly inhibited by vanadate, molybdate, heparin, poly(Glu, Tyr) (4:1) and zinc ions. In order to explore the substrate preferences of this PTPase, we generated 13-residue synthetic phosphotyrosine-containing peptides that corresponded to sites of physiological tyrosine phosphorylation. HPTP beta demonstrated kcat. values between 76 and 258 s-1 using four different phosphopeptides. The substrate preference of HPTP beta was in the order srcTyr-527 > PDGF-RTyr-740 > ERK1Tyr-204 >> CSF-1RTyr-708 with Km values ranging from 140 microM to greater than 10 mM. The variations in affinity were probably due to differences among the four phosphopeptides compared, particularly with respect to the character of the charged amino acids flanking the phosphotyrosine residue.

Here we describe and characterize a small serine/threonine kinase (SSTK) which consists solely of the Nand C-lobes of a protein kinase catalytic domain. SSTK protein is highly conserved among mammals, and no close homologues were found in the genomes of nonmammalian organisms. SSTK specifically interacts with HSP90-1␤, HSC70, and HSP70 proteins, and this association appears to be required for SSTK kinase activity. The SSTK transcript was most abundant in human and mouse testes but was also detected in all human tissues tested. In the mouse testis, SSTK protein was localized to the heads of elongating spermatids. Targeted deletion of the SSTK gene in mice resulted in male sterility due to profound impairment in motility and morphology of spermatozoa. A defect in DNA condensation in SSTK null mutants occurred in elongating spermatids at a step in spermiogenesis coincident with chromatin displacement of histones by transition proteins. SSTK phosphorylated histones H1, H2A, H2AX, and H3 but not H2B or H4 or transition protein 1 in vitro. These results demonstrate that SSTK is required for proper postmeiotic chromatin remodeling and male fertility. Abnormal sperm chromatin condensation is common in sterile men, and our results may provide insight into the molecular mechanisms underlying certain human infertility disorders.Phosphorylation of serine, threonine, and tyrosine residues in substrate targets by protein kinases is a common posttranslational protein modification in eukaryotes and provides a fundamental mechanism for the control of cellular events. Cell division and growth, adhesion and migration, metabolic activity and responses upon environmental stimuli, cell to cell communication, signal transduction, and apoptosis are among the many processes regulated by protein kinases (15,16). At the molecular level, phosphorylation and dephosphorylation of enzymes allow fast and sensitive regulation of enzyme activity and are also a major mechanism of transmembrane signaling and signal amplification in the branching network of intracellular protein kinase cascades that ultimately control gene expression by phosphorylation of transcription factors. Phosphorylation of protein substrates can provide binding sites for protein domains which recognize specific phosphorylated amino acid sequences, thereby mediating protein-protein interactions. Protein kinases constitute a large superfamily of related enzymes which contain 12 conserved subdomains folded into N-and C-lobes of the catalytic domain. The superfamily is subdivided into protein serine/threonine kinases, protein tyrosine kinases, and atypical kinases on the basis of substrate specificity (14,16).Phosphorylation events play a central role in chromatin remodeling during mitosis in somatic cells and during meiosis in mammalian spermiogenesis (2, 10). Much less is known about the mechanisms of postmeiotic chromatin condensation. The structure of chromatin changes dramatically during postmeiotic spermiogenesis, as germ cells develop from round spermatids to fully differ...

The ErbB family of receptors, which include the epidermal growth factor receptor (EGFR), ErbB2, ErbB3, and ErbB4 mediate the actions of a family of bioactive polypeptides. EGF signals through EGFR, whereas heregulin (HRG) signaling is initiated through binding to either ErbB3 or ErbB4. In this report we studied the role of protein-tyrosine phosphatase SHP-2 in ErbB-mediated activation of mitogen-activated protein kinase (MAPK) by overexpressing SHP-2 mutants in COS-7 cells. We demonstrate that enzymatic activity and both NH 2 -and COOH-terminal SH2 domains of SHP-2 are required for EGF-induced MAPK activation, but not for c-Jun amino-terminal kinase stimulation or MAPK activation which occurred in response to myristoylated son of sevenless, activated Ras, or phorbol ester. Dominantnegative forms of SHP-2 had no effect on EGF-stimulated interaction of GRB2 with EGFR or SHC, nor did they influence phosphorylation of SHC and SHC/EGFR association. The same mutant SHP-2 structures that inhibited EGF-mediated stimulation of MAPK also blocked HRG ␣/␤-induced MAPK activation. EGF or HRG ␤ caused SHP-2 SH2 domains to engage multiple phosphotyrosine proteins, and mutation of either domain disrupted these associations. These results demonstrate that SHP-2 performs a common and essential function(s) in ligand-stimulated MAPK activation by the ErbB family of receptors.The ErbB family of receptors, which include epidermal growth factor receptor (EGFR 1 ; ErbB1), ErbB2, ErbB3, and ErbB4, mediate the biological actions of a family of growth factors which are structurally related to EGF (1). This family of bioactive peptides, which includes EGF, transforming growth factor ␣, amphiregulin, heparin-binding EGF-like growth factor, betacellulin, epiregulin, and heregulin ␣/␤ (HRG; neu differentiation factor, neuregulin, acetylcholine receptor-inducing activity, glial growth factor) elicits numerous cellular responses such as mitogenesis, differentiation, trophism, and motility (1). Signaling from ErbBs involves a process of receptor homo-and heterodimerization, which is initiated by engagement of ligand with a specific ErbB receptor (1). EGF and amphiregulin require the presence of EGFR for signaling (2), whereas HRG-induced signal transduction occurs after binding of ligand to either ErbB3 or ErbB4 on cells that co-express ErbB2 (3, 4).The protein-tyrosine phosphatase SHP-2 (PTP1D, SHPTP2, PTP2C, SHPTP3, or Syp) contains two Src homology 2 (SH2) domains (5) and appears to play a critical role in mitogenic responses to EGF and insulin, but not to platelet-derived growth factor (6 -10). Although it is not clear how SHP-2 functions as a positive mediator of EGF signaling, stimulation of cells with EGF has been shown to drive the association of SHP-2 with a number of proteins including a 115-kDa phosphotyrosine (Tyr(P))-containing protein (11), GRB2-associated binder-1 (Gab1) (12), SHP substrate 1 (SHPS-1)/signal-regulatory protein ␣ (SIRP␣) (13,14), and GRB2 via the COOHterminal SH3 domain of GRB2 (15). In Drosophila, membrane targeti...

The epidermal growth factor (EGF) receptor activates several signaling cascades in response to the ligands EGF and amphiregulin (AR). One of these signaling events involves the tyrosine phosphorylation of STATs (signal transducers and activators of transcription), a process believed to require the activation of a tyrosine kinase of the JAK family. In this report we demonstrate that EGF-and AR-induced STAT activation requires the intrinsic kinase activity of the receptor but not the presence of Jak1. We show that both wild type (WT) and truncated EGF receptors lacking all autophosphorylation sites activate STAT 1, 3, and 5 in response to either EGF or AR. Furthermore, relative to cells expressing WT receptor, ligand-induced tyrosine phosphorylation of the STATs was enhanced in cells expressing only the truncated receptor. These results provide the first evidence that (i) EGF receptor-mediated STAT activation occurs in a Jak1-independent manner, (ii) the intrinsic tyrosine kinase activity of the receptor is essential for STAT activation, and (iii) tyrosine phosphorylation sites within the EGF receptor are not required for STAT activation.The activated epidermal growth factor (EGF) 1 receptor mediates a number of important biological responses in mammalian cells including the stimulation of cell division, migration, and differentiation (1). Six different polypeptide ligands, which derive from distinct genes, are capable of binding to the extracellular domain of the EGF receptor. These ligands include EGF, transforming growth factor-␣, amphiregulin (AR), heparin binding EGF-like growth factor, epiregulin, and betacellulin (2). All of these growth factors contain a characteristic EGF-like domain, which is defined by 6 evenly spaced cysteine residues that generate 3 loops through the formation of disulfide bonds. In the case of AR it has been demonstrated that heparan sulfate proteoglycans are necessary for activation of the EGF receptor and a cellular response (3). Upon ligand binding, the intrinsic tyrosine kinase activity of the EGF receptor is augmented, resulting in autophosphorylation as well as the phosphorylation of specific intracellular substrates (4). Among the target proteins that become phosphorylated in response to EGF are mitogen-activated protein kinase, the adapter protein SHC, the GTPase-activating protein of p21 ras , phospholipase C-␥, the phosphotyrosine phosphatase 1D, as well as the non-receptor tyrosine kinase Jak1 (5) and members of the STAT (signal transducers and activators of transcription) family of transcription factors (6 -8). Several reports have shown that the intrinsic receptor kinase activity is necessary for activation of a subset of these proteins, whereas others can be activated also via a kinase-inactive EGF receptor (9 -11). Additional mutations have allowed for the identification of receptor domains that are needed for cell motility, mitogenesis, and activation of mitogen-activated protein kinase or phospholipase C-␥ (12, 13). In the case of the STAT proteins it has been sugges...

The ErbB family of receptors, which includes the epidermal growth factor receptor (EGFR), ErbB2, ErbB3, and ErbB4, mediate signaling by EGF-like polypeptides. To better understand the role of the EGFR tyrosine kinase, we analyzed signaling by a kinase-inactive EGFR (K721M) in ErbB-devoid 32D cells. K721M alone exhibited no detectable signaling capacity, whereas coexpression of K721M with ErbB2, but not ErbB3 or ErbB4, resulted in EGF-dependent mitogen-activated protein kinase (MAPK) activation. The kinase activity, but not tyrosine phosphorylation, of ErbB2 was required for EGF-induced MAPK activation. The presence of tyrosine phosphorylation sites in K721M was not a requisite for signaling, indicating that transphosphorylation of K721M by ErbB2 was not an essential mechanism of receptor activation. Conversely, the mutated kinase domain of K721M (residues 648 -973) and tyrosine phosphorylation of at least one of the receptors were necessary. EGF was found to activate the pro-survival protein kinase Akt in stable cell lines expressing K721M and ErbB2 but, unlike cells expressing wild-type EGFR, was incapable of activating signal transducers and activators of transcription (STAT) or driving cell proliferation. These results demonstrate that EGFR-ErbB2 oligomers are potent activators of MAPK and Akt, and this signaling does not require EGFR kinase activity.