We have raised antibodies against a synthetic dodecapeptide corresponding to the carboxyl terminus of the predicted met gene product. Phosphorylation of 60 kDa and 65 kDa proteins on tyrosine residues was observed when immunoprecipitates of cells containing the activated human met gene were incubated with [Y-~~P]ATP. Phosphoproteins with the same molecular masses could be immunoprecipitated from cells metabolically labelled with [32P]orthophosphate. When considered together, these observations indicate that the activated human met gene encodes 60 kDa and 65 kDa proteins that can catalyse autophosphorylation on tyrosine residues.
Eukaryotic cells contain numerous small-molecular-mass GTP-binding proteins, but the processes that they regulate are not known. Different members of this protein family appear to be associated with specific GTPase-activating proteins (GAPs), and we have previously reported the identification of a cytoplasmic GAP (rho GAP) that stimulates the GTPase activity of p21rho but not of other small-molecular-mass GTP-binding proteins. We have now purified rho GAP 2000-fold from human spleen tissue using f.p.l.c. Electrotransfer of this 27.5 kDa protein on to an Immobilon-P transfer membrane followed by reconstitution of its enzymic activity confirmed its identity. Rho GAP was subjected to N-terminal sequence analysis and 15 amino acids were obtained. The sequence showed 53% identity with a region present in IRA1, a protein which stimulates the GTPase activity of RAS proteins in Saccharomyces cerevisiae. These results suggest that there is a family of sequence-related GAP proteins, which to date includes ras GAP and its yeast counterparts IRA1 and IRA2, rho GAP and the Neurofibromatosis gene product NF1.
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