A Purine Analog‐Sensitive Protein Kinase Activity Associates with Trk Nerve Growth Factor Receptors

Cinzia Volonté, David M. Loeb, Lloyd A. Greene

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

Abstract: 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 ∼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.

Original languageEnglish (US)
Pages (from-to)664-672
Number of pages9
JournalJournal of Neurochemistry
Volume61
Issue number2
DOIs
StatePublished - Jan 1 1993
Externally publishedYes

Fingerprint

Nerve Growth Factor Receptors
Nerve Growth Factor
Protein Kinases
Phosphotransferases
Myelin Basic Protein
Protein-Serine-Threonine Kinases
PC12 Cells
Phosphothreonine
Nerve Growth Factor Receptor
Phosphotyrosine
Phosphorylation
Threonine
Histones
Tyrosine
Association reactions
purine
Substrates
protein kinase N

Keywords

  • 2‐Aminopurine
  • 6‐Thioguanine
  • Nerve growth factor
  • PC12 cells
  • Protein kinase N
  • Trk

ASJC Scopus subject areas

  • Biochemistry
  • Cellular and Molecular Neuroscience

Cite this

A Purine Analog‐Sensitive Protein Kinase Activity Associates with Trk Nerve Growth Factor Receptors. / Volonté, Cinzia; Loeb, David M.; Greene, Lloyd A.

In: Journal of Neurochemistry, Vol. 61, No. 2, 01.01.1993, p. 664-672.

Research output: Contribution to journalArticle

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AB - Abstract: 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 ∼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.

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