PEPTIDE AND PROTEIN-PHOSPHORYLATION BY PROTEIN-TYROSINE KINASE CSK - INSIGHTS INTO SPECIFICITY AND MECHANISM

Csk (C-terminal Src kinase) is a protein tyrosine kinase that phosphor ylates Src family member C-terminal tails, resulting in down-regulatio n of Src family members, The molecular basis of Csk's substrate specif icity and catalytic mechanism with a protein substrate was investigate d, Using a peptide library approach, preferential amino acids which ar e unrelated to the conserved Src C-terminal sequence were identified. The validity of these preferences was confirmed by synthesizing a shor t consensus peptide and demonstrating its high catalytic efficiency wi th Csk. These results underscore the difficulties of relying on amino acids neighboring tyrosine in protein sequences as predictors of prote in kinase substrate specificity for in vivo analysis, In addition, a c atalytically inactive version of the Src family member, Lck (lymphoid cell kinase), was expressed, purified, and evaluated as a Csk substrat e, It was proven to be the most catalytically efficient substrate yet identified for Csk. The high efficiency of purified Csk phosphorylatin g a pure, unphosphorylated Src family member argues against the import ance of an SH2-phosphotyrosine docking interaction or the involvement of extra recruitment proteins in facilitating Csk phosphorylation of S rc family members. Kinetic studies revealed that the chemical step is at least partially rate-determining in Csk-mediated phosphoryl transfe r to the Lck protein, Other properties including preferences for Mn ov er Mg, thio effects, and K-m's for ATP also correlate fairly well betw een protein and peptide phosphorylation. The lack of a significant imp act of increased salt on the K-m for Lck phosphorylation differs from Csk-mediated poly(Glu,Tyr) phosphorylation, and argues against the imp ortance of electrostatic effects in the Csk-Lck binding interaction, T he failure of the Lck phosphorylation product (phosphotyrosine-505) to significantly inhibit Csk phosphorylation of Lck is consistent with a catalytic model involving multidomain structural interactions between substrate and enzyme.