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PMID: 1508179 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

95-kilodalton B-Raf serine/threonine kinase: identification of the protein and its major autophosphorylation site.

Molecular and cellular biology ·Vol. 12 ·No. 9 ·1992-09-00 ·Pages 3733-42

Stephens RM, Sithanandam G, Copeland TD, Kaplan DR, Rapp UR, Morrison DK

Abstract

B-Raf, a member of the Raf family of serine/threonine kinases, is expressed primarily in the brain and in the nervous system. In this study, the biochemical properties of the B-Raf protein were investigated in nerve growth factor (NGF)-responsive cell lines and in brain tissues. B-Raf was identified by using phosphopeptide mapping analysis and cDNA analysis as a 95-kDa protein which is primarily localized in the cytosol. NGF rapidly stimulated both serine and threonine phosphorylation in vivo and autophosphorylation activity in vitro of the B-Raf protein. In PC12 cells, B-Raf autokinase activity was induced by both differentiation factors and mitogens, with maximal activity observed after 5 min of factor addition. B-Raf kinase activity was also observed following NGF treatment of SH-SY5Y neuroblastoma cells and in adult mouse brain and hippocampus. Induction of B-Raf kinase activity in NGF-treated PC12 cells required expression of kinase-active trk receptors. Exogenous substrates or a peptide containing the autophosphorylation site became phosphorylated when added to immune complex kinase assays and reduced the in vitro autophosphorylation activity of B-Raf, suggesting that in vitro autophosphorylation sites and exogenous substrates compete for active sites of the B-Raf kinase. Finally, the major in vitro autophosphorylation site of B-Raf was identified as threonine 372 in the conserved region 2 domain. A threonine residue is present at similar positions in all three mammalian Raf family members and may represent a regulatory site for these proteins.

MeSH Terms
Amino Acid Sequence Animals Blotting, Western Brain/enzymology Enzyme Activation Mice Molecular Sequence Data Nerve Growth Factors/physiology PC12 Cells Peptide Mapping Phosphorylation Protein Kinases/chemistry,genetics,metabolism Protein-Tyrosine Kinases/metabolism Proto-Oncogene Proteins/chemistry,genetics,metabolism Proto-Oncogene Proteins c-raf
Chemicals
Nerve Growth Factors Proto-Oncogene Proteins Protein Kinases Protein-Tyrosine Kinases Proto-Oncogene Proteins c-raf
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Stephens R M
Molecular Mechanisms of Carcinogenesis Laboratory, National Cancer Institute-Frederick Cancer Research and Development Center, Maryland.
Sithanandam G
Copeland T D
Kaplan D R
Rapp U R
Morrison D K
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Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/mcb.12.9.3733-3742.1992
Published
1992-09-00
Pages
3733-42
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC360233
Subset
IM
Grants
NCI NIH HHS · N01-CO-74101 · United States
NCI NIH HHS · N01-CO-74102 · United States
External Links
PubMed source DOI: 10.1128/mcb.12.9.3733-3742.1992 PMC Full Text View journal details
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