The structure of the SHP-2 tyrosine phosphatase, determined at 2.0 Å 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 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 ζ 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, 32â38), 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 Ã resolution, utilizing an active kinase
domain containing residues 327â606 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 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 zeta 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 A 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 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.
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, decavandate is a competitive inhibitor (Ki = 1.8 +/- 0.1 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 366 +/- 10 microM. However, in this case V4+ appears to bind to the Mg(2+)-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 microM, with concentrations above 100 microM being markedly inhibitory. However, even at the optimal 100 microM V4+ concentration, the Vmax and K(m) values (for kemptide) are significantly less favourable than those obtained in the presence of 100 microM Mg2+. In summary, we have found that oxovanadium ions can directly alter the activity of the serine/threonine-specific PKA.
cAMP-dependent protein kinase substrates have been synthesized employing an unusually efficient method that allows the alcohol-bearing residue to be incorporated into the peptide after solid phase ...peptide synthesis. These peptide substrates have been utilized to map the active site substrate specificity of the protein kinase. Only alpha- or beta-substituted alcohol-bearing residues containing the proper absolute configuration are phosphorylated by the enzyme. However, the cAMP-dependent protein kinase will phosphorylate achiral residues. The implications of the observed protein kinase substrate specificity with respect to inhibitor design are discussed.
The substrate specificity of the cAMP-dependent protein kinase has been assessed with peptides bearing threoninol diastereomers.
The threoninol residue contains both a primary alcohol and a secondary ...alcohol, either of which may serve as a site of phosphorylation.
The enzyme-catalyzed phosphorylation of Gly-Arg-Thr-Gly-Arg-Arg-Asn-(2R,3R)-threoninol furnishes a Km of 498 +/- 39 microM
and a Vmax of 7.8 +/- 0.2 mumol/min-mg, whereas the phosphorylation of Gly-Arg-Thr-Gly-Arg-Arg-Asn-(2S,3S)-threoninol provides
a Km of 16.3 +/- 0.8 microM and a Vmax of 16.0 +/- 0.4 mumol/min-mg. Mass spectral analysis of the phosphopeptide reaction
products revealed that each species is phosphorylated only once. 1H-coupled 31P NMR experiments unequivocally demonstrated
that the (2R,3R)-isomer is specifically phosphorylated at the secondary alcohol, whereas the (2S,3S)-isomer is exclusively
phosphorylated at the primary alcohol. This regiospecificity appears to be a consequence of the stereochemistry at C-2 in
the threoninol residues. The structural attributes of the protein kinase that appear to be responsible for the observed differentiation
between the C-2 stereoisomers is discussed.
PDF
