A purine analog-sensitive protein kinase activity associates with Trk nerve growth factor receptors.
Previous studies showed that purine analogs block with varying efficiency and specificity certain effects of nerve growth factor (NGF) on PC12 cells. These compounds also inhibit protein kinase activities. The analog 6-thioguanine has thus far been shown to inhibit only protein kinase N, an NGF-activated protein kinase, whereas 2-aminopurine also blocks other kinases. In the present study, immunoprecipitates of Trk NGF receptors from PC12 cells (+/- NGF treatment) were assayed for protein kinase activity by using the substrates myelin basic protein and histone HF1 under phosphorylating conditions optimal for protein kinase N and in the presence or absence of purine analogs. Activity was detected and approximately 50-80% was inhibited by these compounds. The purine analog-sensitive activity was maximally stimulated by NGF within 5 min, was partially decreased by 10 min, and still remained over basal levels after 15 h of NGF treatment. Analysis of myelin basic protein phosphorylated by anti-Trk immunoprecipitates revealed an NGF-stimulated increase in phosphothreonine and phosphotyrosine. Phosphorylation of threonine, but not of tyrosine residues, was inhibited by 6-thioguanine, which therefore inhibits a serine/threonine kinase associated with NGF receptor rather than the receptor kinase itself. Neither 2-aminopurine 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 with Trk NGF receptors.
Volonté
C
C
Department of Pathology, College of Physicians and Surgeons, Columbia University, New York, New York.
Loeb
D M
DM
Greene
L A
LA
D015075:2-Aminopurine / Q000494:pharmacology; D000818:Animals; D006657:Histones / Q000378:metabolism; D007163:Immunosorbent Techniques; D007700:Kinetics; D004676:Myelin Basic Protein / Q000378:metabolism; D009414:Nerve Growth Factors / Q000494:pharmacology; D016716:PC12 Cells; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D010769:Phosphothreonine / Q000378:metabolism; D011493:Protein Kinase C; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D011687:Purines / Q000494:pharmacology*; D051381:Rats; D020917:Receptor, trkA; D017475:Receptors, Nerve Growth Factor / Q000378:metabolism*; D013866:Thioguanine / Q000494:pharmacology
D006657:Histones; D004676:Myelin Basic Protein; D009414:Nerve Growth Factors; D047428:Protein Kinase Inhibitors; D011518:Proto-Oncogene Proteins; D011687:Purines; D017475:Receptors, Nerve Growth Factor; D010769:Phosphothreonine; D010768:Phosphoserine; D015075:2-Aminopurine; D011494:Protein Kinases; C061570:protein kinase N; D011505:Protein-Tyrosine Kinases; D020917:Receptor, trkA; D011493:Protein Kinase C; D013866:Thioguanine