Synaptic protein tyrosine kinase: partial characterization and identification of endogenous substrates.
The subcellular distribution of protein tyrosine kinase in rat forebrain was determined using [Val5]-angiotensin II as exogenous substrate. Enzyme activity was present in each of the fractions analyzed and was enriched in synaptic membranes (SMs) and the synaptosomal soluble fraction (2.2- and 2.5-fold over the homogenate, respectively). SMs also phosphorylated polyglutamyltyrosine (pGT; molar ratio of 4:1), the Vmax for angiotensin and pGT phosphorylation being 26.3 +/- 1.6 and 142 +/- 4 pmol/min/mg, respectively. Extraction of SMs with several different detergents resulted in enhanced enzyme activity and the solubilization of 33-37% of the angiotensin and 43-70% of the pGT-phosphorylating activity. Isolated postsynaptic densities (PSDs) contained tyrosine kinase and phosphorylated angiotensin and pGT. The Vmax values for angiotensin and pGT phosphorylation by PSDs were 17 +/- 5 and 23 +/- 1 pmol/min/mg, respectively. Six putative endogenous substrates for SM tyrosine kinase, with molecular weights of 205K, 180K, 76K, 60K, 50K, and 45K, were identified. Each of these proteins, except p76, was phosphorylated in the detergent-insoluble residue obtained following the extraction of SMs with Triton X-100 as well as in PSDs, indicating that the postsynaptic apparatus is an active site of tyrosine phosphorylation. The phosphorylation of p76 was localized to the Triton X-100 extract and also occurred in the synaptosomal soluble fraction. The results indicate that tyrosine kinase and its substrates are located in both pre- and postsynaptic compartments and suggest a role for this enzyme in synaptic function.
Ellis
P D
PD
Department of Biochemistry, University of Toronto, Ontario, Canada.
Bissoon
N
N
Gurd
J W
JW
D000804:Angiotensin II / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051381:Rats; D012995:Solubility; D013347:Subcellular Fractions / Q000201:enzymology; D013570:Synaptic Membranes / Q000187:drug effects / Q000201:enzymology*
D000804:Angiotensin II; D011092:Polyethylene Glycols; D008345:Manganese; C031193:angiotensin II, Asp(1)-Val(5)-; D017830:Octoxynol; D011505:Protein-Tyrosine Kinases; D008274:Magnesium