Src homology domains of v-Src stabilize an active conformation of the tyrosine kinase catalytic domain.
To examine the interactions between Src homology domains and the tyrosine kinase catalytic domain of v-Src, various combinations of domains have been expressed in bacteria as fusion proteins. Constructs containing the isolated catalytic domain, SH2 + catalytic domain, and SH3 + SH2 + catalytic domains were active in autophosphorylation assays. For the catalytic domain of v-Src, but not for v-Abl, addition of exogenous Src SH3-SH2 domains stimulated the autophosphorylation activity. In contrast to results for autophosphorylation, constructs containing Src homology domains were more active towards a synthetic peptide substrate than the isolated catalytic domain. The ability of the SH2 and SH3 domains of v-Src to stabilize an active enzyme conformation was also confirmed by refolding after denaturation in guanidinium hydrochloride. Collectively the data suggest that, in addition to their roles in intermolecular protein-protein interactions, the Src homology regions of v-Src exert a positive influence on tyrosine kinase function, potentially by maintaining an active conformation of the catalytic domain.
Xu
B
B
Department of Physiology and Biophysics, School of Medicine, State University of New York at Stony Brook 11794, USA.
Miller
W T
WT
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000378:metabolism; D005982:Glutathione Transferase; D008969:Molecular Sequence Data; D015688:Oncogene Protein pp60(v-src) / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D016374:Oncogene Proteins v-abl / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011487:Protein Conformation; D017510:Protein Folding; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D018909:src Homology Domains
D016374:Oncogene Proteins v-abl; D010455:Peptides; D011993:Recombinant Fusion Proteins; D000255:Adenosine Triphosphate; D005982:Glutathione Transferase; D011505:Protein-Tyrosine Kinases; D015688:Oncogene Protein pp60(v-src)