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Enzymatic characteristics of the c-Raf-1 protein kinase.

Abstract：

:The c-Raf-1 protein kinase plays a central role in the mitogenic response of cells to growth factors, cytokines, and many oncogenes. Despite the critical importance of this enzyme, very little is known of its biochemical properties or mechanisms of regulation. In these experiments, we used the only candidate physiologic substrate identified as yet for c-Raf-1, mitogen-activated protein kinase kinase (MAPKK), to examine enzymatic characteristics and candidate modulators of c-Raf-1, c-Raf-1 was purified from Sf9 cells infected with recombinant baculovirus encoding a histidine-tagged c-Raf-1. The Km values of c-Raf-1 for ATP and MAPKK were 11.6 microM and 0.8 microM, respectively, and the stoichiometry of phosphorylation of MAPKK by c-Raf-1 was 1.67 mol of phosphate per mol of MAPKK. In contrast to prior reports, Mg2+ was the preferred cation at Mg2+ and Mn2+ concentrations > 5 mM. c-Raf-1 substrate specificity was extremely restricted, consistent with the identification of only one candidate physiologic substrate to date and highlighting the necessity of using MAPKK rather than artificial substrates in c-Raf-1 activity assays. Of multiple potential substrates tested, the only one phosphorylated to > 20% of the level of MAPKK phosphorylation was myelin basic protein (22%). Heat-denatured MAPKK was phosphorylated at only 2% the level of native MAPKK, indicating that the restricted substrate specificity may be due to tertiary-structural requirements. We also examined whether c-Raf-1 activity is modulated by lipid binding to the cysteine finger region in its regulatory domain. Of multiple mitogen-stimulated or cell-membrane lipids tested, only phosphatidylserine and diacylglycerol in the presence of Ca2+ (2.5 mM) increased c-Raf-1 kinase activity significantly (1.5-fold). The increase is probably not of physiologic significance because it was about two orders of magnitude less than the stimulation of protein kinase C by these lipids. On gel-filtration chromatography, the peak of c-Raf-1 kinase activity and immunoreactivity eluted at a predicted molecular mass of > 150 kDa, suggesting that active c-Raf-1 (but not inactive c-Raf-1) exists as a multimeric complex. This complex may not include p21ras, however, because immunoreactive p21ras was not identified in the active fractions.

journal_name

Proc Natl Acad Sci U S A

journal_title

Proceedings of the National Academy of Sciences of the United States of America

authors

Force T,Bonventre JV,Heidecker G,Rapp U,Avruch J,Kyriakis JM

doi

10.1073/pnas.91.4.1270

subject

Has Abstract

pub_date

1994-02-15 00:00:00

pages

1270-4

issue

eissn

0027-8424

issn

1091-6490

journal_volume

pub_type

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Enzymatic characteristics of the c-Raf-1 protein kinase.