Protein tyrosine kinase activity and its endogenous substrates in rat brain: a subcellular and regional survey.

Protein phosphorylation has been widely recognized as one of the major regulatory mechanisms in the control of cellular metabolism. Recent studies have provided evidence that protein phosphorylation plays a prominent role in the regulation of neuronal function, including synaptic transmission (Browning et al., 1985; Nairn et al., 1985). Many neurotransmitters and other first messengers regulate the levels of intracellular second messengers such as cyclic AMP, cyclic GMP, calcium, and diacylglycerol, and many of the actions of these second messengers are mediated by the activation of specific classes of serine-and threonine-specific protein kinases (Nestler and Greengard, 1984). Tyrosine-specific protein kinases are a newly discovered class of protein kinases that were originally shown to be involved in the regulation of cell transformation and cell proliferation (for review, see Hunter and Cooper, 1985). Tyrosine-specific protein kinases were first identified as the protein products of the oncogenes of transforming retroviruses (Hunter and Cooper, 1985). Growth factor receptor kinases have been identified as a related class of tyrosine kinases. These include the epidermal growth factor receptor, the insulin receptor, the platelet-derived growth factor receptor, and the insulin-like growth factor receptor (Cohen et al., 1980; Kasuga et al., 1983; Heldin et al., 1983; Jacobs et al., 1983). It appears that the tyrosine-specific protein kinase activity intrinsic to these receptors and to the viral oncogene products mediates many of the effects of these proteins on cellular growth (Hunter and Cooper, 1985). Many of the viral oncogenes have been shown to have normal cellular homologues (for review see Hunter and Cooper, 1985). Recent studies have demonstrated that the gene products of the cellular homologues of the src and yes viral oncogenes are very abundant in nervous tissue, suggesting that protein tyrosine kinases may be involved in the regulation of neuronal function (Barnekow et al., 1982; Cotton and Brugge, 1983; Sorge et al., 1984; Fults et al., 1985; Sudol and Hanafusa, 1986). During the development of the brain, the level of pp60csrc begins to rise after differentiation and remains high in the adult when cell division is very low (Cotton and Brugge, 1983; Sorge et al., 1984; Fults et al., 1985). In addition, recent studies have reported the partial purification and characterization of several protein tyrosine kinases from adult brain (Neer and Lok, 1985; Braun et al., 1986). The presence of high levels of protein tyrosine kinase activity in adult brain, a nonproliferating tissue, suggests that protein tyrosine kinases may play a specific role in the regulation of neuronal function. The nature of this role is as yet unclear. As part of a study of the role of tyrosine-specific protein kinases in neuronal function, we have begun to characterize these kinases in rat brain. In the present investigation, we have studied the tyrosine-specific protein kinase activity in rat CNS using a tyrosine-containing synthetic peptide as exogenous substrate. Synthetic polymers rich in tyrosine and glutamic acid, such as poly(Glu80, Tyr20), have been shown to be effective substrates for various tyrosine-specific protein kinases (Braun et al., 1984). Using this exogenous substrate, we have determined the regional and subcellular distributions of protein tyrosine kinase activity within the rat CNS. We have also characterized the regional and subcellular distribution of the endogenous substrates for the protein tyrosine kinases in rat CNS using phosphotyrosine-specific antibodies.