A PURINE ANALOG SENSITIVE PROTEIN-KINASE ACTIVITY ASSOCIATES WITH TRKNERVE GROWTH-FACTOR RECEPTORS

Previous studies showed that purine analogs block with varying efficie ncy and specificity certain effects of nerve growth factor (NGF) on PC 12 cells. These compounds also inhibit protein kinase activities. The analog 6-thioguanine has thus far been shown to inhibit only protein k inase N, an NGF-activated protein kinase, whereas 2-aminopurine also b locks other kinases. In the present study, immunoprecipitates of Trk N GF receptors from PC12 cells (+/- NGF treatment) were assayed for prot ein kinase activity by using the substrates myelin basic protein and h istone HF1 under phosphorylating conditions optimal for protein kinase N and in the presence or absence of purine analogs. Activity was dete cted and approximately 50-80% was inhibited by these compounds. The pu rine analog-sensitive activity was maximally stimulated by NGF within 5 min, was partially decreased by 10 min, and still remained over basa l levels after 15 h of NGF treatment. Analysis of myelin basic protein phosphorylated by anti-Trk immunoprecipitates revealed an NGF-stimula ted increase in phosphothreonine and phosphotyrosine. Phosphorylation of threonine, but not of tyrosine residues, was inhibited by 6-thiogua nine, which therefore inhibits a serine/threonine kinase associated wi th NGF receptor rather than the receptor kinase itself. Neither 2-amin opurine nor 6-thioguanine inhibited the NGF-dependent induction of Trk -associated kinase activity. Our findings thus indicate association of a purine analog-sensitive serine/threonine protein kinase activity wi th Trk NGF receptors.