Characterization of pp60c-src Tyrosine Kinase Activities Using a Continuous Assay: Autoactivation of the Enzyme Is an Intermolecular Autophosphorylation Process

Abstract

A continuous assay for pp60c-src tyrosine kinase (srcTK) was developed. A lag in phosphorylation of the peptide RRLIEDAEYAARG was observed that could be eliminated by preincubation with MgATP. The induction time for this lag was dependent upon MgATP and srcTK concentrations. When autophosphorylation was monitored by 32P incorporation from [gamma-32P]ATP, a lag in the time course was also observed. These results demonstrate that autoactivation is an intermolecular process. The electrospray ionization mass spectrum of the enzyme before and after activation demonstrated an increase in the phosphorylation state of the enzyme after incubation with MgATP. The delta 85-N-terminal mutant protein and a full-length G2A pp60c-src mutant, which removes the myristylation site, used in these studies were partially phosphorylated on Y338 and Y530 as isolated. This is the first report of phosphorylation on Y338, but the significance of this site of phosphorylation is unknown. These phosphorylations were insufficient to active the enzyme for transfer of the gamma-phosphoryl of MgATP to the peptides. The unphosphorylated enzyme initially present was converted to a monophosphorylated species upon treatment with MgATP. Y-419 phosphorylation was evident only after treatment with MgATP. These data are consistent with autophosphorylation on Y-419 as predicted. Intermolecular autophosphorylation is consistent with the ability of srcTK to dimerize, which is analogous to activation of receptor tyrosine kinases such as the EGF receptor kinase in response to growth factors. These results indicate that dimerization leading to activation does not require binding to the membrane or a hydrophobic N-terminus in the case of srcTK.

Autophosphorylation of Src and Yes blocks their inactivation by Csk phosphorylation

“…This result further demonstrates that the lack of time-dependent complete inactivation of Yes Ya by Csk is the result of autophosphorylation. Furthermore, these results indicate that the autophosphorylation that blocks Csk inactivation is on the main autophosphorylation site within the activation loop of Yes and Src, although other sites have been reported to also undergo autophosphorylation in the Src family (Osusky et al, 1995;Barker et al, 1995;Jullien et al, 1994). This result also indicates that autophosphorylation will likely have the same function in modulating Csk regulation of other PTKs in the Src family.…”

The Activated Form of the Lck Tyrosine Protein Kinase in Cells Exposed to Hydrogen Peroxide Is Phosphorylated at Both Tyr-394 and Tyr-505

“…Our results agree with data presented by others who showed that the Src tyrosine kinase retains activity when phosphorylated on Tyr-416 and Tyr-527 (41). 3 Previous work in our laboratory as well as kinetic data by other groups suggest that the activating phosphorylation of Tyr-394 in Src family members is an intermolecular event rather than intramolecular reaction (22,(42)(43)(44). 4 Intermolecular phosphorylation of Tyr-394 may be carried out by Lck in vivo, but it is quite possible that other Src family members, or non-Src tyrosine kinases, may also act to phosphorylate Tyr-394 and activate Lck.…”

Variant estrogen receptor–c-Src molecular interdependence and c-Src structural requirements for endothelial NO synthase activation

“…The availability of a low level of c-Src kinase activity in resting cells (13) and the increasing association of ER46 with E2 stimulation (Fig. 3A) may favor homotypic (26,27) and heterotypic (24,25) protein interactions with c-Src and further enforce c-Src ''opening.'' Indeed, it has been demonstrated that doubly phosphorylated c-Src can be active, such that Y419 phosphorylation can override the p-Y530-directed inhibition (28).…”