CATALYTIC ACTIVITY OF THE SH2 DOMAIN OF HUMAN PP6O(C-SRC-CENTER-DOT),EVIDENCE FROM NMR, MASS-SPECTROMETRY, SITE-DIRECTED MUTAGENESIS AND KINETIC-STUDIES FOR AN INHERENT PHOSPHATASE-ACTIVITY

During solution structural studies it was apparent that the human reco mbinant pp60(c-src) SH2 domain (srcSH2, residues 144-249) possessed an inherent phosphatase (Pase) activity. Complexes of U[C-13,N-15]srcSH2 with unlabeled Ac-pYEEIE ((I) under bar) were examined using P-31 and H-1-detected isotope filtered NMR methods. The presence of a high-aff inity complex in equimolar solutions of (I) under bar and U[C-13, N-15 ]-srcSH2 was demonstrated by chemical shift perturbations, line broade ning, and the observation of intermolecular nuclear Overhauser effects from the pY and lie side-chain protons of (I) under bar to protons on amino acid residues present in the binding pocket of srcSH2. Solution s containing excess (I) under bar relative to srcSH2 revealed a slow h ydrolysis of (I) under bar to produce Ac-YEEIE and inorganic phosphate . The hydrolysis rate determined from NMR and HPLC-electrospray ioniza tion mass spectrometry data at 30 degrees C for solutions containing e xcess (I) under bar was 0.002-0.003 h(-1). srcSH2 also catalyzed the h ydrolysis of p-nitrophenyl phosphate (pNPP). Isoelectric focusing gels of a number of mutant srcSH2s demonstrated that this activity comigra ted with srcSH2. K-m, k(cat), and k(cat)/K-m were 3.7 +/- 0.4 mM, 3.1 +/- 0.2 x 10(-2) min(-1), and 8.4 +/- 0.4 M(-1) min(-1), respectively, toward pNPP. The C188A mutant of the srcSH2 domain displayed 15% of t he activity displayed by wild-type srcSH2, demonstrating that this res idue is not absolutely required for activity. Two additional mutations in the known pY binding site, R178K and R158K, also resulted in decre ased pNPPase activity, suggesting that the activity resides in or near this site. The inhibitor profile and pH dependence suggest that this is a novel protein Pase,activity. Other than phosphate (competitive in hibitor, K-i=50 mu M), the activity was not inhibited by known inhibit ors of Ser/Thr or Tyr protein kinases. Inhibitors of Ser/Thr Pases wer e also not inhibitory, but the pNPPase activity was inhibited by the p rotein tyrosine phosphatase inhibitor orthovanadate. pY-containing pep tides inhibited the pNPPase activity, but the potency did not parallel the apparent binding affinity to the SH2 site. The data are consisten t with a catalytically active form of SH2 that is present in small amo unts. These data suggest that srcSH2 may play a catalytic role in sign al transduction and regulation of pp60(c-src) activity.