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Enzymatic characteristics of the c-Raf-1 protein kinase
T Force1, J V Bonventre, G Heidecker
1Cardiac Unit, Massachusetts General Hospital, Boston 02114.
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.
Insights
This study characterizes the biochemical properties of c-Raf-1 protein kinase, revealing its strict substrate specificity for mitogen-activated protein kinase kinase (MAPKK) and identifying phosphatidylserine and diacylglycerol as weak modulators. Active c-Raf-1 appears to exist as a multimeric complex.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Signaling
Background:
- c-Raf-1 protein kinase is crucial for cellular responses to growth factors and oncogenes.
- Its biochemical properties and regulatory mechanisms remain largely uncharacterized.
- Mitogen-activated protein kinase kinase (MAPKK) is the only identified physiological substrate for c-Raf-1.
Purpose of the Study:
- To investigate the enzymatic characteristics of c-Raf-1.
- To identify potential modulators of c-Raf-1 activity.
- To explore the quaternary structure of active c-Raf-1.
Main Methods:
- Purification of histidine-tagged c-Raf-1 from Sf9 cells using recombinant baculovirus.
- Enzymatic assays using MAPKK as the substrate to determine kinetic parameters (Km) and cation preference.
- Analysis of substrate specificity using various potential substrates.
- Investigation of lipid modulation of c-Raf-1 activity.
- Gel-filtration chromatography to assess the molecular mass and complex formation of active c-Raf-1.
Main Results:
- Determined Km values for ATP (11.6 microM) and MAPKK (0.8 microM).
- Established phosphorylation stoichiometry of 1.67 mol phosphate/mol MAPKK.
- Identified Mg2+ as the preferred cation over Mn2+ at concentrations > 5 mM.
- Demonstrated highly restricted substrate specificity, with myelin basic protein being the only other minor substrate.
- Showed that heat-denatured MAPKK is poorly phosphorylated, indicating tertiary structure dependence.
- Observed a 1.5-fold increase in c-Raf-1 activity with phosphatidylserine and diacylglycerol in the presence of Ca2+, but this effect was physiologically insignificant compared to Protein Kinase C.
- Found that active c-Raf-1 elutes at > 150 kDa, suggesting a multimeric complex, which does not appear to include p21ras.
Conclusions:
- c-Raf-1 exhibits strict substrate specificity for MAPKK, dependent on tertiary structure.
- While certain lipids can weakly modulate activity, their physiological relevance is questionable.
- Active c-Raf-1 likely exists as a multimeric complex, distinct from p21ras.