Tyrosine Protein Kinases in Membrane Fractions from Rat Cerebral Cortex1

Abstract

Specific activities of tyrosine tubulin kinase in the particulate fractions from rat cerebellum, medulla oblongata, hypothalamus, striatum, midbrain, and cerebral cortex ranged within 30% of each other and more than 3 times higher than those in the soluble fractions. In the cerebral cortex, tyrosine protein kinase activity toward tubulin and tyrosine-glutamate (1:4) copolymers was mainly distributed in the plasma membrane and the microsome fractions. The kinase activity in cerebral cortex particulate fractions was quantitatively solubilized and separated into two peaks, kinase I and kinase II, by Sephacryl S-300 gel filtration in the presence of 0.2% Nonidet P-40 and 0.2 M NaCl. Kinases I and II were each resolved into 5 active peaks (I-1----5 and II-1----5) by casein-Sepharose column chromatography. The molecular weights of these kinases were estimated from the s20,w values to be 59,000-65,000. The Km values of II-1----5 for tubulin were nearly 10 times higher than those of I-1----5. However, the Km values of the two groups of kinases for tyrosine-glutamate copolymers were not so significantly different. About 60% of the copolymers kinase activity in I-3, I-4, II-3, and II-4 was immunoprecipitable with a saturating amount of monoclonal antibody against pp60c-src. Incubation of the immunoprecipitates with ATP resulted in the autophosphorylation of a 60 kDa protein in I-3 and I-4, and a 52 kDa protein in II-3 and II-4. Immunoblotting also indicated I-3 and I-4 as 60 kDa bands and II-3 and II-4 as 52 kDa bands on SDS-polyacrylamide gels.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein Tyrosine Kinases in Human Brain and Gliomas

“…In these controls the specific activity in the membrane fraction was more than three times higher than in the cytosolic fraction. Previously, comparable results were obtained for rat brain (Tsukamoto et al, 1988;Kobayashi et al, 1989).…”

“…Multiple autocrine loops involving receptors with associated PTK activities are present in human gliomas (Gammeltoft et al, 1988;Nister et al, 1988;Westphal and Hermann, 1989). In addition, it has been reported that the expression of c-src, yes, and fyn is high in normal human brain (Sudol and Hanafusa, 1986;Yamanashi et al, 1987;Tsukamoto et al, 1988). These findings prompted us to compare total PTK activities in normal brain and gliomas using a recently developed nonradioactive dot-blot assay .…”

“…This raises the question of the origin of these kinases. It cannot be excluded that some members of the src family of membrane-bound PTKs are localized in the cytosol as well (Krueger et al, 1980;Tsukamoto et al, 1988). To investigate the identity of the cytosolic PTKs, immunoprecipitation and immunoblotting experiments were performed using an antibody [clone 443 (Lipsich et al, 1983) This antibody immunoprecipitated -30% of the cytosolic activity of both normal brain and gliomas and recognized M, 60,000 protein bands.…”

“…In normal human brain high levels of the prototype of the membrane-bound PTKs, the c-src protein, are expressed (Okado and Nakagawa, 1988;Tsukamoto et al, 1988). It has been shown that the specific activity of the c-src protein from neuronal cultures is six to 12 times higher than in astrocyte cultures (Brugge et al,1 985).…”