95-KILODALTON B-RAF SERINE THREONINE KINASE - IDENTIFICATION OF THE PROTEIN AND ITS MAJOR AUTOPHOSPHORYLATION SITE

被引:76
作者
STEPHENS, RM
SITHANANDAM, G
COPELAND, TD
KAPLAN, DR
RAPP, UR
MORRISON, DK
机构
[1] NCI, FREDERICK CANC RES & DEV CTR, MOLEC MECH CARCINOGENESIS LAB, POB B, FREDERICK, MD 21702 USA
[2] NCI, FREDERICK CANC RES & DEV CTR, ABL BASIC RES PROGRAM, PROT STRUCT GRP, FREDERICK, MD 21702 USA
[3] NCI, VIRAL CARCINOGENESIS LAB, FREDERICK, MD 21702 USA
[4] PROGRAM RESOURCES INC DYNCORP, BIOL CARCINOGENESIS & DEV PROGRAM, FREDERICK, MD 21702 USA
关键词
D O I
10.1128/MCB.12.9.3733
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
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页码:3733 / 3742
页数:10
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