The structure of the SHP-2 tyrosine phosphatase, determined at 2.0 angstroms resolution, shows how its catalytic activity is regulated by its two SH2 domains. In the absence of a tyrosine-phosphorylated binding partner, the N-terminal SH2 domain binds the phosphatase domain and directly blocks its active site. This interaction alters the structure of the N-SH2 domain, disrupting its phosphopeptide-binding cleft. Conversely, interaction of the N-SH2 domain with phosphopeptide disrupts its phosphatase recognition surface. Thus, the N-SH2 domain is a conformational switch; it either binds and inhibits the phosphatase, or it binds phosphoproteins and activates the enzyme. Recognition of bisphosphorylated ligands by the tandem SH2 domains is an integral element of this switch; the C-terminal SH2 domain contributes binding energy and specificity, but it does not have a direct role in activation.

Src homology 2 (SH2) domains are phosphotyrosine binding modules found within many cytoplasmic proteins. A major function of SH2 domains is to bring about the physical assembly of signaling complexes. We now show that, in addition, simultaneous occupancy of both SH2 domains of the phosphotyrosine phosphatase SH-PTP2 (Syp, PTP 1D, PTP-2C) by a tethered peptide with two IRS-1-derived phosphorylation sites potently stimulates phosphatase activity. The concentration required for activation by the tethered peptide is 80-160-fold lower than either corresponding monophosphorylated peptide. Moreover, the diphosphorylated peptide stimulates catalytic activity 37-fold, compared with 9-16-fold for the monophosphorylated peptides. Mutational analyses of the SH2 domains of SH-PTP2 confirm that both SH2 domains participate in this effect. Binding studies with a tandem construct comprising the N- plus C-terminal SH2 domains show that the diphosphorylated peptide binds with 60-90-fold higher affinity than either monophosphorylated sequence. These results demonstrate that SH-PTP2 activity can be potently regulated by interacting via both of its SH2 domains with phosphoproteins having two cognate phosphorylation sites.

The domain organization of many signalling proteins facilitates a segregation of binding, catalytic and regulatory functions. The mammalian SH2 domain protein tyrosine phosphatases (PTPs) contain tandem SH2 domains and a single carboxy-terminal catalytic domain. SH-PTP1 (PTP1C, HCP) and SH-PTP2 (Syp, PTP2C, PTP1D) function downstream from tyrosine kinase-linked insulin, growth factor, cytokine and antigen receptors. As well as directing subcellular localization by binding to receptors and their substrates, the two SH2 domains of these PTPs function together to regulate catalysis. Here we report the structure of the tandem SH2 domains of SH-PTP2 in complex with monophosphopeptides. A fixed relative orientation of the two domains, stabilized by a disulphide bond and a small hydrophobic patch within the interface, separates the peptide binding sites by approximately 40 A. The defined orientation of the SH2 domains in the structure, and data showing that peptide orientation and spacing between binding sites is critical for enzymatic activation, suggest that spatial constraints are important in this multidomain protein-protein interaction.

In Xenopus ectodermal explants (animal caps), fibroblast growth factor (FGF) evokes two major events: induction of ventrolateral mesodermal tissues and elongation. The Xenopus FGF receptor (XFGFR) and certain downstream components of the XFGFR signal transduction pathway (e.g., members of the Ras/Raf/MEK/ mitogen-activated protein kinase [MAPK] cascade) are required for both of these processes. Likewise, activated versions of these signaling components induce mesoderm and promote animal cap elongation. Previously, using a dominant negative mutant approach, we showed that the protein-tyrosine phosphatase SHP-2 is necessary for FGF-induced MAPK activation, mesoderm induction, and elongation of animal caps. Taking advantage of recent structural information, we now have generated novel, activated mutants of SHP-2. Here, we show that expression of these mutants induces animal cap elongation to an extent comparable to that evoked by FGF. Surprisingly, however, activated mutant-induced elongation can occur without mesodermal cytodifferentiation and is accompanied by minimal activation of the MAPK pathway and mesodermal marker expression. Our results implicate SHP-2 in a pathway(s) directing cell movements in vivo and identify potential downstream components of this pathway. Our activated mutants also may be useful for determining the specific functions of SHP-2 in other signaling systems.Growth factor-mediated signal transduction is a well-recognized mechanism for controlling cell growth, differentiation, and movement. Orchestration of these processes is essential for complex events such as embryonic development. Studies of the African clawed frog, Xenopus laevis, have shown that members of the transforming growth factor ␤ and fibroblast growth factor (FGF) families are essential for mesodermal tissue differentiation and for coordinating cell movements associated with gastrulation (22,49,51). Both of these processes can be studied ex vivo in Xenopus animal caps (ectodermal explants). Stimulation of animal caps with activin (a transforming growth factor ␤ family member) induces a wide range of tissues, including (depending on the dose) dorsal and ventral mesoderm, neural tissue, and endoderm. The induced dorsal mesoderm undergoes convergence and extension movements (reviewed in reference 51), which drive dramatic elongation of the animal caps. In embryos, these movements provide the primary motive force for gastrulation. FGF family members induce a more restricted range of tissues in animal caps, including ventrolateral mesodermal derivatives such as muscle, mesenchyme, and mesothelium, but do not induce neural tissue, endoderm, or dorsal mesodermal structures, such as notochord (24). FGF stimulation also causes shape changes in animal caps, but FGF-induced elongation is distinct from that evoked by activin. It has been assumed that one or more of the tissues induced by FGF drive FGF-stimulated elongation. However, it has not been shown explicitly that FGF-stimulated elongation depends on FGF-induced differentiation. Ad...

The ZAP-70 tyrosine kinase plays a critical role in T cell activation and the immune response and therefore is a logical target for immunomodulatory therapies. Although the crystal structure of the tandem Src homology-2 domains of human ZAP-70 in complex with a peptide derived from the subunit of the T cell receptor has been reported (Hatada, M. H., Lu, X., Laird, E. R., Green, J., Morgenstern, J. P., Lou, M., Marr, C. S., Phillips, T. B., Ram, M. K., Theriault, K., Zoller, M. J., and Karas, J. L. (1995) Nature 377, 32-38), the structure of the kinase domain has been elusive to date. We crystallized and determined the three-dimensional structure of the catalytic subunit of ZAP-70 as a complex with staurosporine to 2.3 Å resolution, utilizing an active kinase domain containing residues 327-606 identified by systematic N-and C-terminal truncations. The crystal structure shows that this ZAP-70 kinase domain is in an active-like conformation despite the lack of tyrosine phosphorylation in the activation loop. The unique features of the ATP-binding site, identified by structural and sequence comparison with other kinases, will be useful in the design of ZAP-70-selective inhibitors.The zeta-associated protein, 70 kDa (ZAP-70), 1 a Syk family tyrosine kinase associated with the subunit of the T cell receptor, is primarily expressed in T and NK cells and plays an essential role in signaling through the T cell receptor (TCR) (2, 3). TCR-mediated activation of T cells is crucial to the immune response. Transplant rejection and diseases, such as allergic responses and autoimmune disorders, stem from a failure to adequately modulate T cell activation. In humans, ZAP-70 gene mutations have been identified that confer lower ZAP-70 protein expression levels or catalytically inactive ZAP-70 proteins (4 -6). ZAP-70 deficiency results in the absence of mature CD8 ϩ T cells and the prevention of TCR-mediated activation of CD4 ϩ T cells, and it can lead to severe combined immunodeficiency. Peptides that block the association of ZAP-70 with the subunit (7,8) and peptides that antagonize ZAP-70 tyrosine kinase activity (9) block T cell activation in vitro. These studies indicate that ZAP-70 antagonists could be useful immunomodulatory therapeutic agents.ZAP-70 contains two N-terminal SH2 (Src homology domain 2) domains and a C-terminal kinase domain. During T cell activation, the binding of ZAP-70 SH2 domains to the phosphorylated subunit on the activated TCR complex causes a colocalization with the Lck tyrosine kinase that phosphorylates ZAP-70 on Tyr 493 in the activation loop (10, 11). Also, ZAP-70 autophosphorylates multiple tyrosines in the region between the SH2 domains and the kinase domain (10, 12), including the binding sites for additional SH2-containing signaling proteins such as SLP-76, Lat, Lck, PLC␥1, Vav, Shc, Ras-GAP, and Abl (reviewed in Refs. 8, 13, and 15). ZAP-70-mediated activation of these downstream effectors leads to the release of intracellular calcium stores, and the transcription of interleukin-2 and other ...

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Vanadium oxoions have been shown to elicit a wide range of effects in biological systems, including an increase in the quantity of phosphorylated proteins. This response has been attributed to the inhibition of protein phosphatases, the indirect activation of protein kinases via stimulation of enzymes at early steps in signal transduction pathways and/or the direct activation of protein kinases. We have evaluated the latter possibility by exploring the effects of vanadate, decavanadate and vanadyl cation species on the activity of the cAMP-dependent protein kinase (PKA), a serine/threonine kinase. Vanadate, in the form of monomer, dimer, tetramer and pentamer species, neither inhibits nor activates PKA. In marked contrast, decavanadate is a competitive inhibitor (Ki = 1.8ŷ0.1 mM) of kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly), a peptide-based substrate. This inhibition pattern is especially surprising, since the negatively charged decavanadate would not be predicted to bind to the region of the active site of the enzyme that accommodates the positively charged kemptide substrate. Our studies suggest that decavanadate can associate with kemptide in solution, which would prevent kemptide from interacting with the enzyme. Vanadium(IV) also inhibits the PKA-catalysed phosphorylation of kemptide, but with an IC50 of 366ŷ10 ƁM. However, in this case V4+ appears to bind to the Mg2+-binding site, since it can substitute for Mg2+. In the absence of Mg2+, the optimal concentration of vanadium(IV) for the PKA-catalysed phosphorylation of kemptide is 100 ƁM, with concentrations above 100 ƁM being markedly inhibitory. However, even at the optimal 100 ƁM V4+ concentration, the Vmax and Km values (for kemptide) are significantly less favourable than those obtained in the presence of 100 ƁM Mg2+. In summary, we have found that oxovanadium ions can directly alter the activity of the serine/threonine-specific PKA.

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