The Product of the Raf Oncogene

Raf is a 74 kDa serine/threonine kinase that is activated in vivo by an unknown mechanism (as of early 1999!). Inactive Raf is brought to the membrane by active GTP-Ras.. Raf then is activated and phosphorylates MEK., the only known in vivo target of Raf (Morrison and Cutler,1997). At the membrane, RasGTP has some effect on Raf kinase activity through the Raf zinc finger (Roy et al., 1997; Luo et al., 1997), but the majority of Raf activation at the membrane is by unknown mechanisms and is an active area of investigation.

Raf has essentially two domains, an N-terminal negative regulatory domain and a C-terminal catalytic domain (Cutler et al., 1998). There are at least three isoforms, A-, B- and C-Raf. Each of the isoforms varies in its basal kinase activity and ability to be activated by different molecules (Marais et al., 1997). Of particular interest for neurologists is B-Raf, the predominant form of Raf in neural cells, which is activated by cAMP, directly by protein kinase A phosphorylation and by Rap binding in PC12 cells (Vossler et al., 1997).

In the cytosol, inactive Raf is in a complex with 14-3-3 proteins and some heat shock proteins. While the role of 14-3-3 is controversial, some consensus is appearing, 14-3-3 may be required for Raf stability by crosslinking two Rafs together as a dimer which can be activated (Tzivion et al., 1998) and 14-3-3 may also be required for the efficient cycling of Raf on and off the membrane (Roy et al., 1998). Other important molecules in the Raf activation process include KSR, which may act as scaffold to deliver MEK to Raf (Denoul-Galy et al., 1998; Yu et al., 1998), phospholipids, which may help to stabilise Raf (Ghosh et al., 1996) and caveolin, a scaffolding protein which may sequester inactive Raf and lots of other signalling proteins (Schlegel et al., 1998).

Some kinases phosphorylate Raf, including Protein Kinase C (Marais et al., 1998), Protein Kinase A, which also phosphorylates the Ras effector, RalGDS (Kikuchi and Williams, 1996), Pak3, which may link Raf to G-protein coupled signalling (King et al., 1998) and SRC (Stokoe and McCormick, 1997). The physiological relevance of thesephosphorylations remains to be determined.

The activation of Raf at the plasma membrane is not a simple one-step process. Whether dimerisation, lipid binding or kinase/phosphatase is responsible for activation in vivo, it WILL be interesting.

Kindly contributed by: Rob McPherson

References

• Cutler RE Jr, Stephens RM, Saracino MR, Morrison DK Proc Natl Acad Sci U S A 1998 Aug 4;95(16):9214-9219 Autoregulation of the Raf-1 serine/threonine kinase.
• Denouel-Galy A, Douville EM, Warne PH, Papin C, Laugier D, Calothy G, Downward J, Eychene A Curr Biol 1998 Jan 1;8(1):46-55 Murine Ksr interacts with MEK and inhibits Ras-induced transformation.
• Ghosh S, Strum JC, Sciorra VA, Daniel L, Bell RM J Biol Chem 1996 Apr 5;271(14):8472-80 Raf-1 kinase possesses distinct binding domains for phosphatidylserine and phosphatidic acid. Phosphatidic acid regulates the translocation of Raf-1 in 12-O-tetradecanoylphorbol-13-acetate-stimulated Madin-Darby canine kidney cells.
• Kikuchi A, Williams LT J Biol Chem 1996 Jan 5;271(1):588-94 Regulation of interaction of ras p21 with RalGDS and Raf-1 by cyclic AMP-dependent protein kinase.
• King AJ, Sun H, Diaz B, Barnard D, Miao W, Bagrodia S, Marshall MS Nature 1998 Nov 12;396(6707):180-183 The protein kinase Pak3 positively regulates Raf-1 activity through phosphorylation of serine 338.
• Luo Z, Diaz B, Marshall MS, Avruch J Mol Cell Biol 1997 Jan;17(1):46-53 An intact Raf zinc finger is required for optimal binding to processed Ras and for ras-dependent Raf activation in situ.
• Marais R, Light Y, Paterson HF, Mason CS, Marshall CJ J Biol Chem 1997 Feb 14;272(7):4378-4383 Differential regulation of Raf-1, A-Raf, and B-Raf by oncogenic ras and tyrosine kinases.
• Marais R, Light Y, Mason C, Paterson H, Olson MF, Marshall CJ Science 1998 Apr 3;280(5360):109-112 Requirement of Ras-GTP-Raf complexes for activation of Raf-1 by protein kinase C.
• Morrison DK, Cutler RE Curr Opin Cell Biol 1997 Apr;9(2):174-179 The complexity of Raf-1 regulation.
• Roy S, Lane A, Yan J, McPherson R, Hancock JF J Biol Chem 1997 Aug 8;272(32):20139-45 Activity of plasma membrane-recruited Raf-1 is regulated by Ras via the Raf zinc finger.
• Roy S, McPherson RA, Apolloni A, Yan J, Lane A, Clyde-Smith J, Hancock JF Mol Cell Biol 1998 Jul;18(7):3947-3955 14-3-3 facilitates Ras-dependent Raf-1 activation in vitro and in vivo.
• Schlegel A, Volonte D, Engelman JA, Galbiati F, Mehta P, Zhang XL, Scherer PE, Lisanti MP Cell Signal 1998 Jul;10(7):457-463 Crowded little caves: structure and function of caveolae.
• Stokoe D, McCormick F EMBO J 1997 May 1;16(9):2384-96 Activation of c-Raf-1 by Ras and Src through different mechanisms: activation in vivo and in vitro.
• Tzivion G, Luo Z, Avruch J Nature 1998 Jul 2;394(6688):88-92 A dimeric 14-3-3 protein is an essential cofactor for Raf kinase activity.
• Vossler MR, Yao H, York RD, Pan MG, Rim CS, Stork PJ Cell 1997 Apr 4;89(1):73-82 cAMP activates MAP kinase and Elk-1 through a B-Raf- and
• Rap1-dependent pathway.
• Yu W, Fantl WJ, Harrowe G, Williams LT Curr Biol 1998 Jan 1;8(1):56-64 Regulation of the MAP kinase pathway by mammalian Ksr through direct interaction with MEK and ERK.