Raf serine/threonine kinase

Figure 3. MAP kinase regulatory pathway. The MAP kinase signaling pathway begins with activation of the receptor tyrosine kinase (RTK) by exogenous signals, such as growth factors and insulin. The signal is then transmitted into the cell via activation of the Raf serine/threonine kinase either directly by the RTK or through the GTP-binding protein, Ras. The signal is then transmitted to the nucleus and to other cytoplasmic proteins via MAPKK and MAPK.

At least 10 distinct families of Ras effector proteins have been identified, with the Raf serine/threonine kinases, phos-phatidylinositol 3-kinases (PI3K), and RalGEFs being the most extensively characterized (3) (see Fig. 4). Beyond their shared ability to bind preferentially to Ras-GTP, Ras effectors possess highly divergent biochemical and biologic functions. 

The final link in the kinase cascade activated by Ras-GTP emerged from studies in which scientists fractionated extracts of cultured cells searching for a kinase activity that could phosphorylate MAP kinase and that was present only in cells stimulated with growth factors, not quiescent cells. This work led to Identification of MEK, a kinase that specifically phosphorylates one threonine and one tyrosine residue on MAP kinase, thereby activating its catalytic activity. (The acronym MEK comes from MAP and ERK kinase.) Later studies showed that MEK binds to the C-terminal catalytic domain of Raf and is phosphorylated by the Raf serine/ threonine kinase this phosphorylation activates the catalytic activity of MEK. Hence, activation of Ras induces a kinase cascade that Includes Raf, MEK, and MAP kinase activated RTK Ras Raf MEK MAP kinase. 

The antisense oligonucleotide LErafAON against the serine/threonine kinase c-Raf has been tested in phase I clinical trials. The antisense oligonucleotides ISIS-5132, which also inhibits c-Raf, and ISIS-3521, which inhibits PKC, went through different phase clinical trials with solid tumour patients. Unfortunately, no objective responses occurred with these PKI. GEM-231, an oligonucleotide targeting the RIa subunit of protein kinase A is currently undergoing phase I/II clinical trials alone or in combination with traditional therapy for the treatment of solid cancers [3]. 

Sorafenib is a multitargeted cancer therapy that inhibits VEGFR, PDGFR, KIT, fetal liver tyrosine kinase 3 (FLT-3), and the serine/threonine kinase RAF. RAF kinase is a key downstream effector of Ras in the MAPK/Ras signal-transduction pathway that has been linked to various cancers. Sorafenib is both a tyrosine kinase inhibitor and serine/threonine signal-transduction inhibitor. Sorafenib has been approved in renal cancer. 

Polymorphism of the MC1R gene on chromosome 16 (16q24), which is associated with red hair, also increases susceptibility to melanoma.23 Mutations affecting the serine-threonine kinase B-RAF gene have been reported with high rates in individuals with MM.24 The RAF serine-threonine... 

B-raf, a member of the raf gene family of serine/threonine kinases, is expressed as two major transcripts of 4.0 kb and 2.6 kb in the mouse testis (Wadewitz et al., 1993). B-raf expression is limited to the germ cells and is particularly abundant in early spermatids. Northern hybridization analysis revealed that the two B-ra/transcripts are expressed in a stage-specific manner. Low levels of the 4.0-kb transcript are first... 

Morrison, D, K., Kaplan, D. R Escobedo, J. A., Rapp, U Roberts, T. M., and Williams, L. T. (1989). Direct activation of the serine/threonine kinase activity of Raf-1 through tyrosine phosphorylation by the PDGFP-receptor. Cell 58 649-657. 

CREB is also phosphorylated on serine 133 by stimulation of growth factor signaling cascades [63]. This occurs via a complex pathway involving MAPK cascades (Fig. 23-9). Thus, as outlined earlier, nerve growth factor and related neurotrophins that act on receptor tyrosine kinases lead to the successive activation of Ras, Raf, MEK and ERK. Activated ERK then phosphorylates and activates a serine-threonine kinase, RSK, particular subtypes of which directly activate CREB via the phosphorylation of serine 133. 

There are at least three major effector pathways that are activated by neurotrophic factor-Trk receptors. The best-characterized pathway is the extracellular-regulated kinase (ERK) cascade, which is regifiated by activation of Ras, a small membrane-bound G protein. Activation of Ras occurs when activated Trk receptor associates with adaptor proteins and a GTP exchange factor (see Russell and Duman 2002 for details). Ras in turn recruits and activates a serine threonine kinase, Raf, to the membrane resulting in the activation of ERK kinase (also referred to as MEK) and ERK (also known as mitogen activated protein kinase or MAPK). Activation of the Ras-Raf-MEK-ERK cascade can lead to regifiation of many celMar proteins, including ribosomal S6-kinase (RSK). 

Guan KL, Figueroa C, Brtva TR, Zhu T, Taylor J, Barber TD, Vojtek AB (2000) Negative regulation of the serine/threonine kinase B-Raf by Akt. J Biol Chem 275(35) 27354-27359... 

Vojtek, A. B., Hollenberg, S. M., and Cooper, J. A. (1993) Mammalian Ras interacts directly with the serine/threonine kinase Raf. Cell 74,205-214. 

Sorafenib (l)11 is a multikinase inhibitor marketed by Bayer and Onyx. Sorafenib blocks tyrosine kinases as well as serine/threonine kinases. Its story began in 1994 when Bayer and Onyx entered a collaboration to discover novel Raf/MEK/ERK inhibitors. They first discovered a very mildly active compound 8 (/C50 17 pM) against Rafl kinase in 1995 from screening a collection of 200,000 compounds. The optimization of its potency and its ADMET profile using medicinal chemistry and combinatorial chemistry methods led to the identification of sorafenib (1) in 1999 as a preclinical development candidate. Multiple phase I studies started in 2000, when sorafenib tosylate (19) was evaluated in patents with advanced solid tumors of different types. In December 2005, Sorafenib tosylate (19) received U.S. FDA approval for the treatment of advanced renal cell carcinoma (RCC). Two years later, it was approved for the treatment of unresectable hepatocellular carcinoma (HCC). 

It has been shown that the Raf-1 protein serine/threonine kinase is a druggable signaling molecule in cancer therapy (5). and Kasid and co-workers have developed cationic liposomes for in vivo delivery of raf antisense oligonucleotides and raf siRNA into human tumor xenografts established in immunodeficient mice. The authors provide very useful protocols for the preparation of a modified cationic liposome/antisense oligonucleotide formulation as well as toxicology, pharmacokinetics, biodistribution, and anti-tumor efficacy studies in mice (5). 

We and others have demonstrated that Raf-1 protein serine/threonine kinase is a druggable signaling molecule in cancer therapy (1,13,17,21-25). Our laboratory has developed a novel cationic liposomal formulation for systemic delivery of intact raf ASO (LErafAON) to normal and tumor tissues in mice (13,17). The liposome-entrapped raf antisense oligonucleotide (LErafAON) is also the first liposomal ASO drug tested in humans (26,27). Systemically delivered cationic liposomal nanoparticles containing rafsiRNA (LErafsiRNA) also inhibit Raf-1 protein expression in tumor and most normal tissues in human prostate tumor (PC-3)-bearing athymic mice (Fig. 1 and Color Plate 1, see Color Plate Section). 

Signalling by G-protein-coupled receptors is terminated by binding of the receptor kinase to the receptor, followed by phosphorylation. Receptor kinases are serine/threonine kinases, recognizing the active conformation of the receptor. A carrier function is attributed to Py-subunits in bringing the kinase to the receptor in the membrane. 25 For that function, isoprenylation of the Py-complex is required. This function is comparable to that of Ras, bringing the cytosolic Raf kinase to the membrane for activation. Both the y-subunits of the Py-complex and Ras are modified by prenylation (see Chapter 3). Control of G proteins by RGS proteins... 

RAF s are serine/threonine kinases, encoded by protooncogens. They share common structural properties with proteinkinase C, both in their conserved C-terminal catalytic... 

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