Uncoupling Raf1 from MEK1/2 Impairs Only a Subset of Cellular Responses to Raf Activation

Abstract

The Raf family of serine/threonine protein kinases is intimately involved in the transmission of cell regulatory signals controlling proliferation and differentiation. The best characterized Raf substrates are MEK1 and MEK2. The activation of MEK1/2 by Raf is required to mediate many of the cellular responses to Raf activation, suggesting that MEK1/2 are the dominant Raf effector proteins. However, accumulating evidence suggests that there are additional Raf substrates and that subsets of Raf-induced regulatory events are mediated independently of Raf activation of MEK1/2. To examine the possibility that there is bifurcation at the level of Raf in activation of MEK1/2-dependent and MEK1/2-independent cell regulatory events, we engineered a kinase-active Raf1 variant (RafBXB(T481A)) with an amino acid substitution that disrupts MEK1 binding. We find that disruption of MEK1/2 association uncouples Raf from activation of ERK1/2, induction of serum-response element-dependent gene expression, and induction of growth and morphological transformation. However, activation of NF-κB-dependent gene expression and induction of neurite differentiation were unimpaired. In addition, Raf-dependent activation of p90 ribosomal S6 kinase was only slightly impaired. These results support the hypothesis that Raf kinases utilize multiple downstream effectors to regulate distinct cellular activities. The Raf family of serine/threonine protein kinases is intimately involved in the transmission of cell regulatory signals controlling proliferation and differentiation. The best characterized Raf substrates are MEK1 and MEK2. The activation of MEK1/2 by Raf is required to mediate many of the cellular responses to Raf activation, suggesting that MEK1/2 are the dominant Raf effector proteins. However, accumulating evidence suggests that there are additional Raf substrates and that subsets of Raf-induced regulatory events are mediated independently of Raf activation of MEK1/2. To examine the possibility that there is bifurcation at the level of Raf in activation of MEK1/2-dependent and MEK1/2-independent cell regulatory events, we engineered a kinase-active Raf1 variant (RafBXB(T481A)) with an amino acid substitution that disrupts MEK1 binding. We find that disruption of MEK1/2 association uncouples Raf from activation of ERK1/2, induction of serum-response element-dependent gene expression, and induction of growth and morphological transformation. However, activation of NF-κB-dependent gene expression and induction of neurite differentiation were unimpaired. In addition, Raf-dependent activation of p90 ribosomal S6 kinase was only slightly impaired. These results support the hypothesis that Raf kinases utilize multiple downstream effectors to regulate distinct cellular activities. mitogen-activated protein kinase/extracellular signal-regulated kinase kinase mitogen-activated protein extracellular ligand-regulated kinases nuclear factor κB ribosomal S6 kinase luciferase green fluorescent protein heparin-binding epidermal growth factor-like growth factor hemagglutinin glutathione S-transferase human embryonic kidney serum-response element Cellular interpretation of growth regulatory signals requires functional grouping of molecules into signal transduction cascades. The Raf family of serine/threonine protein kinases is critically involved in this signal transduction process. Raf kinases were first discovered as gain of function mutants with the ability to induce growth and morphological transformation of established cell lines. Subsequently it was discovered that activation of cellular Raf proteins is a downstream response to growth factors and is required to link growth factor receptor signaling to activation of gene expression (1Daum G. Eisenmann-Tappe I. Fries H.-W. Troppmair J. Rapp U.R. Trends Biochem. Sci. 1994; 19: 474-480Abstract Full Text PDF PubMed Scopus (483) Google Scholar). Studies of genetic model systems have demonstrated that activation of Raf is an essential step in many growth and developmental programs, and studies of tumor model systems have demonstrated that Raf mediates transformation by many oncogenes (2Naumann U. Eisenmann-Tappe I. Rapp U.R. Recent Res. Cancer Res. 1997; 147: 237-244Crossref Scopus (41) Google Scholar).A major substrate of activated Raf is mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK1).1 Upon activation by Raf, MEK1 can in turn phosphorylate and activate the p42 and p44 MAP kinases (also known as extracellular ligand-regulated kinases, ERK1 and 2) (1Daum G. Eisenmann-Tappe I. Fries H.-W. Troppmair J. Rapp U.R. Trends Biochem. Sci. 1994; 19: 474-480Abstract Full Text PDF PubMed Scopus (483) Google Scholar). Activated ERKs phosphorylate a number of cytoplasmic and nuclear targets, including transcription factors that mediate growth factor regulation of gene expression. ERK activation is required for cellular transformation induced by oncogenic Ras and Raf (3Khosravi-Far R. Solski P.A. Kinch M.S. Burridge K. Der C.J. Mol. Cell. Biol. 1995; 13: 6443-6453Crossref Scopus (639) Google Scholar), and it has been reported that expression of constitutively activated MEK1 is sufficient to induce cellular transformation of immortalized fibroblasts (4Cowley S. Paterson H. Kemp P. Marshall C.J. Cell. 1994; 77: 841-852Abstract Full Text PDF PubMed Scopus (1845) Google Scholar, 5Mansour S.J. Matten T.W. Hermann A.S. Candia J.M. Rong S. Fukasawa K. Vande Woude G.F. Ahn N.G. Science. 1994; 265: 966-970Crossref PubMed Scopus (1254) Google Scholar).These studies suggest that activation of MEK1, with subsequent ERK activation, is the primary event mediating cellular responses to activated Raf. However, several cellular responses to activated Raf have been characterized that appear to be independent of MEK or MAP kinase activation. For example, constitutively active variants of Raf but not MEK are sufficient to induce differentiation of hippocampal neuronal cells (6Kuo W.L. Abe M. Rhee J. Eves E.M. McCarthy S.A. Yan M. Templeton D.J. McMahon M. Rosner M.R. Mol. Cell. Biol. 1996; 16: 1458-1470Crossref PubMed Scopus (79) Google Scholar). Expression of mutationally activated Raf1 in CCL 39 cells has been reported to result in an ERK1/2-independent activation of p70 S6 kinase, leading to increased translation of mRNAs with polypyrimidine tracts (7Lenormand P. McMahon M. Pouyssegur J. J. Biol. Chem. 1996; 271: 15762-15768Crossref PubMed Scopus (81) Google Scholar). Raf-mediated activation of NF-κB transcription factors may occur independently of MEK1/2 activation in Jurkat T cells (8Baumann B. Weber C.K. Troppmair J. Whiteside S. Israel A. Rapp U.R. Wirth T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4615-4620Crossref PubMed Scopus (154) Google Scholar), and Raf activity promotes, whereas MEK1/2 activity inhibits, atrial natriuretic factor expression in cardiac myocytes (9Jette C. Thorburn A. FEBS Lett. 2000; 467: 1-6Crossref PubMed Scopus (13) Google Scholar). Consistent with these observations, candidate Raf substrates in addition to MEK1 have been reported (10Galaktionov K. Jessus C. Beach D. Genes Dev. 1995; 9: 1046-1058Crossref PubMed Scopus (230) Google Scholar, 11Wang H.G. Rapp U.R. Reed J.C. Cell. 1996; 87: 629-638Abstract Full Text Full Text PDF PubMed Scopus (706) Google Scholar, 12Lin J.H. Makris A. McMahon C. Bear S.E. Patriotis C. Prasad V.R. Brent R. Golemis E.A. Tsichlis P.N. J. Biol. Chem. 1999; 274: 14706-14715Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar).To further explore the potential of Raf kinases to modulate cell regulatory pathways independently of MEK1/2 activation, we isolated a kinase active, MEK1/2 binding-defective Raf variant, RafBXB(T481A). RafBXB(T481A) can efficiently stimulate morphological changes in PC12 cells indicative of differentiation events, NF-κB-dependent gene expression, and activation of p90 ribosomal S6 kinase (RSK), despite severely impaired ERK1/2 activation.DISCUSSIONAlthough it is clear that MEK1 and MEK2 are major substrates of Raf, accumulating observations suggest that Raf may regulate important cellular events independently of, or in parallel with, MEK1/2 activation (see the Introduction). To begin to explore this concept in detail, we have engineered a kinase-active Raf1 variant that is uncoupled from MEK1/2-dependent signaling events because of an amino acid substitution (T481A of human Raf1) that interferes with MEK1/2 binding. Expression of this Raf variant (RafBXB(T481A)) in cells revealed that it is uncoupled from activation of ERK1 and ERK2, cannot induce growth and morphological transformation, and cannot activate transcription from serum response elements or the HB-EGF promoter. In contrast RafBXB(T481A) retained the ability to induce neurite-like outgrowths in PC12 cells, activate transcription from NF-κB-dependent promoters, and induce the kinase activity of p90 RSK.The above results suggest that RafBXB(T481A) can be used to define subsets of Raf-induced responses that are MEK-independent. Our observation that Raf activation of NF-κB can be uncoupled from MEK1/2 regulation is consistent with recent data examining NF-κB regulation in Jurkat T cells (8Baumann B. Weber C.K. Troppmair J. Whiteside S. Israel A. Rapp U.R. Wirth T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4615-4620Crossref PubMed Scopus (154) Google Scholar). Our observations that RafBXB(T481A) mimics RafBXB effects on PC12 cell neurite differentiation and p90 RSK activation were more unexpected. Experiments using dominant inhibitory variants of MEK1 and the pharmacological inhibitor PD98059 suggest that MEK activation is required for Raf-induced neurite differentiation and p90 RSK activation (4Cowley S. Paterson H. Kemp P. Marshall C.J. Cell. 1994; 77: 841-852Abstract Full Text PDF PubMed Scopus (1845) Google Scholar, 21Schouten G.J. Vertegaal A.C. Whiteside S.T. Israel A. Toebes M. Dorsman J.C. van der Eb A.J. Zantema A. EMBO J. 1997; 16: 3133-3144Crossref PubMed Scopus (205) Google Scholar, 22Ghoda L. Lin X. Greene W.C. J. Biol. Chem. 1997; 272: 21281-21288Crossref PubMed Scopus (181) Google Scholar, 23Pang L. Sawada T. Decker S.J. Saltiel A.R. J. Biol. Chem. 1995; 270: 13585-13588Abstract Full Text Full Text PDF PubMed Scopus (895) Google Scholar, 24Alessi D.R. Cuenda A. Cohen P. Dudley D.T. Saltiel A.R. J. Biol. Chem. 1995; 270: 27489-27494Abstract Full Text Full Text PDF PubMed Scopus (3246) Google Scholar). Our results suggest that although some extent of MEK1/2 activation may be required for these responses, activated Raf can contribute to neurite differentiation and RSK activation independently of MEK1/2 activation. It is important to note that dominant interfering MEK1/2 variants retain the ability to associate with Raf and therefore may block both MEK1/2-dependent and MEK1/2-independent Raf functions (25Janssen R.A. Veenstra K.G. Jonasch P. Jonasch E. Mier J.W. J. Biol. Chem. 1998; 273: 32182-32186Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). In addition, both of the widely used chemical inhibitors of MEK1/2, PD98059 and U0126, also inhibit at least one other MEK family member involved with Raf signaling, MEK5 (26Kamakura S. Moriguchi T. Nishida E. J. Biol. Chem. 1999; 274: 26563-26571Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar).The epistatic relationships of proteins functioning in mammalian signal transduction cascades are most often explored using combinations of constitutively active and dominant interfering variants of the components that contribute to regulation of the cascade. This approach has been highly successful in ordering the components of the Raf/MEK/ERK protein kinase cascade and for assessing the importance of this cascade in mediating mitogenic signals (27English J. Pearson G. Wilsbacher J. Swantek J. Karandikar M. Xu S. Cobb M.H. Exp. Cell Res. 1999; 253: 255-270Crossref PubMed Scopus (377) Google Scholar). However, as a more sophisticated understanding of the molecular mechanisms of cell regulation develops, it is becoming clear that many signal transduction proteins are multifunctional, introducing branch-points into what were once considered to be simple linear pathways of information flow (28Pawson T. Scott J.D. Science. 1997; 278: 2075-2080Crossref PubMed Scopus (1887) Google Scholar,29Zuker C.S. Ranganathan R. Science. 1999; 283: 650-651Crossref PubMed Scopus (26) Google Scholar). In the context of complex regulatory networks, phenotypes observed with dominant interfering variants of signaling proteins can often be difficult to interpret because of association of the variants with multiple regulatory and effector proteins.As all of the components that mediate Raf action in cells have not been identified, we currently can not rule out the formal possibility that RafBXB(T481A) regulation of neurite induction, and p90 RSK is mediated by activation of ERK1/2 to that are the of the of RafBXB(T481A) from ERK1/2 activation, activation, and is in contrast to the regulation of neurite and p90 RSK activation. of the of action of RafBXB(T481A) in cells, as as the of additional Raf variants that association with contribute to a understanding of the of cellular Raf kinase Cellular interpretation of growth regulatory signals requires functional grouping of molecules into signal transduction cascades. The Raf family of serine/threonine protein kinases is critically involved in this signal transduction process. Raf kinases were first discovered as gain of function mutants with the ability to induce growth and morphological transformation of established cell lines. Subsequently it was discovered that activation of cellular Raf proteins is a downstream response to growth factors and is required to link growth factor receptor signaling to activation of gene expression (1Daum G. Eisenmann-Tappe I. Fries H.-W. Troppmair J. Rapp U.R. Trends Biochem. Sci. 1994; 19: 474-480Abstract Full Text PDF PubMed Scopus (483) Google Scholar). Studies of genetic model systems have demonstrated that activation of Raf is an essential step in many growth and developmental programs, and studies of tumor model systems have demonstrated that Raf mediates transformation by many oncogenes (2Naumann U. Eisenmann-Tappe I. Rapp U.R. Recent Res. Cancer Res. 1997; 147: 237-244Crossref Scopus (41) Google Scholar). major substrate of activated Raf is mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK1).1 Upon activation by Raf, MEK1 can in turn phosphorylate and activate the p42 and p44 MAP kinases (also known as extracellular ligand-regulated kinases, ERK1 and 2) (1Daum G. Eisenmann-Tappe I. Fries H.-W. Troppmair J. Rapp U.R. Trends Biochem. Sci. 1994; 19: 474-480Abstract Full Text PDF PubMed Scopus (483) Google Scholar). Activated ERKs phosphorylate a number of cytoplasmic and nuclear targets, including transcription factors that mediate growth factor regulation of gene expression. ERK activation is required for cellular transformation induced by oncogenic Ras and Raf (3Khosravi-Far R. Solski P.A. Kinch M.S. Burridge K. Der C.J. Mol. Cell. Biol. 1995; 13: 6443-6453Crossref Scopus (639) Google Scholar), and it has been reported that expression of constitutively activated MEK1 is sufficient to induce cellular transformation of immortalized fibroblasts (4Cowley S. Paterson H. Kemp P. Marshall C.J. Cell. 1994; 77: 841-852Abstract Full Text PDF PubMed Scopus (1845) Google Scholar, 5Mansour S.J. Matten T.W. Hermann A.S. Candia J.M. Rong S. Fukasawa K. Vande Woude G.F. Ahn N.G. Science. 1994; 265: 966-970Crossref PubMed Scopus (1254) Google Scholar). These studies suggest that activation of MEK1, with subsequent ERK activation, is the primary event mediating cellular responses to activated Raf. However, several cellular responses to activated Raf have been characterized that appear to be independent of MEK or MAP kinase activation. For example, constitutively active variants of Raf but not MEK are sufficient to induce differentiation of hippocampal neuronal cells (6Kuo W.L. Abe M. Rhee J. Eves E.M. McCarthy S.A. Yan M. Templeton D.J. McMahon M. Rosner M.R. Mol. Cell. Biol. 1996; 16: 1458-1470Crossref PubMed Scopus (79) Google Scholar). Expression of mutationally activated Raf1 in CCL 39 cells has been reported to result in an ERK1/2-independent activation of p70 S6 kinase, leading to increased translation of mRNAs with polypyrimidine tracts (7Lenormand P. McMahon M. Pouyssegur J. J. Biol. Chem. 1996; 271: 15762-15768Crossref PubMed Scopus (81) Google Scholar). Raf-mediated activation of NF-κB transcription factors may occur independently of MEK1/2 activation in Jurkat T cells (8Baumann B. Weber C.K. Troppmair J. Whiteside S. Israel A. Rapp U.R. Wirth T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4615-4620Crossref PubMed Scopus (154) Google Scholar), and Raf activity promotes, whereas MEK1/2 activity inhibits, atrial natriuretic factor expression in cardiac myocytes (9Jette C. Thorburn A. FEBS Lett. 2000; 467: 1-6Crossref PubMed Scopus (13) Google Scholar). Consistent with these observations, candidate Raf substrates in addition to MEK1 have been reported (10Galaktionov K. Jessus C. Beach D. Genes Dev. 1995; 9: 1046-1058Crossref PubMed Scopus (230) Google Scholar, 11Wang H.G. Rapp U.R. Reed J.C. Cell. 1996; 87: 629-638Abstract Full Text Full Text PDF PubMed Scopus (706) Google Scholar, 12Lin J.H. Makris A. McMahon C. Bear S.E. Patriotis C. Prasad V.R. Brent R. Golemis E.A. Tsichlis P.N. J. Biol. Chem. 1999; 274: 14706-14715Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). To further explore the potential of Raf kinases to modulate cell regulatory pathways independently of MEK1/2 activation, we isolated a kinase active, MEK1/2 binding-defective Raf variant, RafBXB(T481A). RafBXB(T481A) can efficiently stimulate morphological changes in PC12 cells indicative of differentiation events, NF-κB-dependent gene expression, and activation of p90 ribosomal S6 kinase (RSK), despite severely impaired ERK1/2 activation. it is clear that MEK1 and MEK2 are major substrates of Raf, accumulating observations suggest that Raf may regulate important cellular events independently of, or in parallel with, MEK1/2 activation (see the Introduction). To begin to explore this concept in detail, we have engineered a kinase-active Raf1 variant that is uncoupled from MEK1/2-dependent signaling events because of an amino acid substitution (T481A of human Raf1) that interferes with MEK1/2 binding. Expression of this Raf variant (RafBXB(T481A)) in cells revealed that it is uncoupled from activation of ERK1 and ERK2, cannot induce growth and morphological transformation, and cannot activate transcription from serum response elements or the HB-EGF promoter. In contrast RafBXB(T481A) retained the ability to induce neurite-like outgrowths in PC12 cells, activate transcription from NF-κB-dependent promoters, and induce the kinase activity of p90 RSK.The above results suggest that RafBXB(T481A) can be used to define subsets of Raf-induced responses that are MEK-independent. Our observation that Raf activation of NF-κB can be uncoupled from MEK1/2 regulation is consistent with recent data examining NF-κB regulation in Jurkat T cells (8Baumann B. Weber C.K. Troppmair J. Whiteside S. Israel A. Rapp U.R. Wirth T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4615-4620Crossref PubMed Scopus (154) Google Scholar). Our observations that RafBXB(T481A) mimics RafBXB effects on PC12 cell neurite differentiation and p90 RSK activation were more unexpected. Experiments using dominant inhibitory variants of MEK1 and the pharmacological inhibitor PD98059 suggest that MEK activation is required for Raf-induced neurite differentiation and p90 RSK activation (4Cowley S. Paterson H. Kemp P. Marshall C.J. Cell. 1994; 77: 841-852Abstract Full Text PDF PubMed Scopus (1845) Google Scholar, 21Schouten G.J. Vertegaal A.C. Whiteside S.T. Israel A. Toebes M. Dorsman J.C. van der Eb A.J. Zantema A. EMBO J. 1997; 16: 3133-3144Crossref PubMed Scopus (205) Google Scholar, 22Ghoda L. Lin X. Greene W.C. J. Biol. Chem. 1997; 272: 21281-21288Crossref PubMed Scopus (181) Google Scholar, 23Pang L. Sawada T. Decker S.J. Saltiel A.R. J. Biol. Chem. 1995; 270: 13585-13588Abstract Full Text Full Text PDF PubMed Scopus (895) Google Scholar, 24Alessi D.R. Cuenda A. Cohen P. Dudley D.T. Saltiel A.R. J. Biol. Chem. 1995; 270: 27489-27494Abstract Full Text Full Text PDF PubMed Scopus (3246) Google Scholar). Our results suggest that although some extent of MEK1/2 activation may be required for these responses, activated Raf can contribute to neurite differentiation and RSK activation independently of MEK1/2 activation. It is important to note that dominant interfering MEK1/2 variants retain the ability to associate with Raf and therefore may block both MEK1/2-dependent and MEK1/2-independent Raf functions (25Janssen R.A. Veenstra K.G. Jonasch P. Jonasch E. Mier J.W. J. Biol. Chem. 1998; 273: 32182-32186Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). In addition, both of the widely used chemical inhibitors of MEK1/2, PD98059 and U0126, also inhibit at least one other MEK family member involved with Raf signaling, MEK5 (26Kamakura S. Moriguchi T. Nishida E. J. Biol. Chem. 1999; 274: 26563-26571Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar).The epistatic relationships of proteins functioning in mammalian signal transduction cascades are most often explored using combinations of constitutively active and dominant interfering variants of the components that contribute to regulation of the cascade. This approach has been highly successful in ordering the components of the Raf/MEK/ERK protein kinase cascade and for assessing the importance of this cascade in mediating mitogenic signals (27English J. Pearson G. Wilsbacher J. Swantek J. Karandikar M. Xu S. Cobb M.H. Exp. Cell Res. 1999; 253: 255-270Crossref PubMed Scopus (377) Google Scholar). However, as a more sophisticated understanding of the molecular mechanisms of cell regulation develops, it is becoming clear that many signal transduction proteins are multifunctional, introducing branch-points into what were once considered to be simple linear pathways of information flow (28Pawson T. Scott J.D. Science. 1997; 278: 2075-2080Crossref PubMed Scopus (1887) Google Scholar,29Zuker C.S. Ranganathan R. Science. 1999; 283: 650-651Crossref PubMed Scopus (26) Google Scholar). In the context of complex regulatory networks, phenotypes observed with dominant interfering variants of signaling proteins can often be difficult to interpret because of association of the variants with multiple regulatory and effector proteins.As all of the components that mediate Raf action in cells have not been identified, we currently can not rule out the formal possibility that RafBXB(T481A) regulation of neurite induction, and p90 RSK is mediated by activation of ERK1/2 to that are the of the of RafBXB(T481A) from ERK1/2 activation, activation, and is in contrast to the regulation of neurite and p90 RSK activation. of the of action of RafBXB(T481A) in cells, as as the of additional Raf variants that association with contribute to a understanding of the of cellular Raf kinase it is clear that MEK1 and MEK2 are major substrates of Raf, accumulating observations suggest that Raf may regulate important cellular events independently of, or in parallel with, MEK1/2 activation (see the Introduction). To begin to explore this concept in detail, we have engineered a kinase-active Raf1 variant that is uncoupled from MEK1/2-dependent signaling events because of an amino acid substitution (T481A of human Raf1) that interferes with MEK1/2 binding. Expression of this Raf variant (RafBXB(T481A)) in cells revealed that it is uncoupled from activation of ERK1 and ERK2, cannot induce growth and morphological transformation, and cannot activate transcription from serum response elements or the HB-EGF promoter. In contrast RafBXB(T481A) retained the ability to induce neurite-like outgrowths in PC12 cells, activate transcription from NF-κB-dependent promoters, and induce the kinase activity of p90 The above results suggest that RafBXB(T481A) can be used to define subsets of Raf-induced responses that are MEK-independent. Our observation that Raf activation of NF-κB can be uncoupled from MEK1/2 regulation is consistent with recent data examining NF-κB regulation in Jurkat T cells (8Baumann B. Weber C.K. Troppmair J. Whiteside S. Israel A. Rapp U.R. Wirth T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4615-4620Crossref PubMed Scopus (154) Google Scholar). Our observations that RafBXB(T481A) mimics RafBXB effects on PC12 cell neurite differentiation and p90 RSK activation were more unexpected. Experiments using dominant inhibitory variants of MEK1 and the pharmacological inhibitor PD98059 suggest that MEK activation is required for Raf-induced neurite differentiation and p90 RSK activation (4Cowley S. Paterson H. Kemp P. Marshall C.J. Cell. 1994; 77: 841-852Abstract Full Text PDF PubMed Scopus (1845) Google Scholar, 21Schouten G.J. Vertegaal A.C. Whiteside S.T. Israel A. Toebes M. Dorsman J.C. van der Eb A.J. Zantema A. EMBO J. 1997; 16: 3133-3144Crossref PubMed Scopus (205) Google Scholar, 22Ghoda L. Lin X. Greene W.C. J. Biol. Chem. 1997; 272: 21281-21288Crossref PubMed Scopus (181) Google Scholar, 23Pang L. Sawada T. Decker S.J. Saltiel A.R. J. Biol. Chem. 1995; 270: 13585-13588Abstract Full Text Full Text PDF PubMed Scopus (895) Google Scholar, 24Alessi D.R. Cuenda A. Cohen P. Dudley D.T. Saltiel A.R. J. Biol. Chem. 1995; 270: 27489-27494Abstract Full Text Full Text PDF PubMed Scopus (3246) Google Scholar). Our results suggest that although some extent of MEK1/2 activation may be required for these responses, activated Raf can contribute to neurite differentiation and RSK activation independently of MEK1/2 activation. It is important to note that dominant interfering MEK1/2 variants retain the ability to associate with Raf and therefore may block both MEK1/2-dependent and MEK1/2-independent Raf functions (25Janssen R.A. Veenstra K.G. Jonasch P. Jonasch E. Mier J.W. J. Biol. Chem. 1998; 273: 32182-32186Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). In addition, both of the widely used chemical inhibitors of MEK1/2, PD98059 and U0126, also inhibit at least one other MEK family member involved with Raf signaling, MEK5 (26Kamakura S. Moriguchi T. Nishida E. J. Biol. Chem. 1999; 274: 26563-26571Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar). The epistatic relationships of proteins functioning in mammalian signal transduction cascades are most often explored using combinations of constitutively active and dominant interfering variants of the components that contribute to regulation of the cascade. This approach has been highly successful in ordering the components of the Raf/MEK/ERK protein kinase cascade and for assessing the importance of this cascade in mediating mitogenic signals (27English J. Pearson G. Wilsbacher J. Swantek J. Karandikar M. Xu S. Cobb M.H. Exp. Cell Res. 1999; 253: 255-270Crossref PubMed Scopus (377) Google Scholar). However, as a more sophisticated understanding of the molecular mechanisms of cell regulation develops, it is becoming clear that many signal transduction proteins are multifunctional, introducing branch-points into what were once considered to be simple linear pathways of information flow (28Pawson T. Scott J.D. Science. 1997; 278: 2075-2080Crossref PubMed Scopus (1887) Google Scholar,29Zuker C.S. Ranganathan R. Science. 1999; 283: 650-651Crossref PubMed Scopus (26) Google Scholar). In the context of complex regulatory networks, phenotypes observed with dominant interfering variants of signaling proteins can often be difficult to interpret because of association of the variants with multiple regulatory and effector proteins. all of the components that mediate Raf action in cells have not been identified, we currently can not rule out the formal possibility that RafBXB(T481A) regulation of neurite induction, and p90 RSK is mediated by activation of ERK1/2 to that are the of the of RafBXB(T481A) from ERK1/2 activation, activation, and is in contrast to the regulation of neurite and p90 RSK activation. of the of action of RafBXB(T481A) in cells, as as the of additional Raf variants that association with contribute to a understanding of the of cellular Raf kinase