THE ROLE OF Raf-1 PHOSPHORYMTION IN SIGNAL TRANSDUCTION Gisela Heidecker,* Walter Kolch,*1 Deborah K. Morrison,t and Ulf R. Rapp* 'Viral Pathology Section, Laboratory of Viral Carcinogenesis, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland 21702,
tABL- Basic Research Program, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland 21702
I. The raf Oncogene Family Raf- 1 Activation following Growth Factor Stimulation A. Modes of Raf-1 Activation B. Raf-1 Activation in Cells Expressing Oncogenically Activated PTKs 111. Sites of Raf- 1 Phosphorylation A. Sites of Serine/Threonine Phosphorylation B. Sites of Tyrosine Phosphorylation I v. Consequences of Raf-1 Activation A. Raf- 1 in Activated Receptor Complexes B. Relative Position of Raf in the Signaling Cascade C. Substrates of Activated Raf-1 V. Conclusion References 11.
1. The raf Oncogene Family Raf proteins are serine/threonine-specific protein kinases that function in signal transduction, transmitting mitogenic signals from the ligand-activated growth factor receptors at the cell surface to the transcriptional machinery in the nucleus. The general properties of raffamily genes and their role in carcinogenesis have recently been reviewed (Rapp, 1991; Kolch et al., 1990a; Storm et al., 1990a). Briefly, so far three active genes, c-raf-1, A-raf-1, and B-raf have been described in human, mouse, rat, and chicken cells. In mouse and chicken cells (in the latter, the gene is called mil), truncated versions of c-raf-1 and B-raf have been found as oncogenes of acutely transforming viruses (Rapp et al., 1983a,b; Jansen et al., 1983, 1984; Sutrave et al., 1984; Evchene et al., 1990), and all three genes can be oncogenically activated in vitro (Huleihe1 et al., 1986; Heidecker et al., 1990; Sithanandam et al., 1990). They have been mapped to three different chromosomes and are located at Current address: W e c k e AG, Biological Research and Biotechnology, Mooswaldalleel-9, 7800 Freiburg, Germany.
53 ADVANCES IN CANCER RESEARCH, VOL. 58
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
54
GISELA HEIDECKER ET AL.
sites that are frequently altered in human tumors (Sithanandam et al., 1989; Storm et al., 1990a,b). c-ruf-1 is ubiquitously expressed although at variable levels. A-ruf transcripts are most prominent in urogenital tissues and B-raf expression is most abundant in testis and cerebrum (Storm et al., 1990b). T h e raf genes, like most oncogenes, are highly conserved, and homologues have been identified in Xenopur (Le Guellec et al., 1988) and Drosophila (Mark and Rapp, 1984; Mark et al., 1987), in which one of the two gene family members was first identified as the developmental l(1) polehole gene (Ambrosio et al., 1989) acting in concert with the torso gene, which ,encodes a transmembrane phosphotyrosine kinase. A raf-related gene has recently been isolated from CaenorhabditG elegans (Georgi et al., 1990), which combines a Raf-like kinase domain with an amino-terminal region with similarity to the transmembrane and receptor part of transmembrane receptor kinases found in higher organisms. Based on phylogenetic studies, the raf genes, together with mos, encode the only serine/threonine kinases that belong to the src superfamily, which otherwise encodes protein tyrosine kinases (PTKs) (Hanks et al., 1988). 11. Raf-1 Activation following Growth Factor Stimulation
In this article we will concentrate on the evidence for entry of cytosolic Raf- 1 kinase into activated receptor complexes as well as complexdependent or -independent phosphorylation of Raf- 1 on tyrosine and serine residues, which leads to enzyme activation. So far, very little work in this area has addressed the roles of A- o r B-raJ T h e involvement of Raf-1 has been analyzed in more than a dozen receptor systems, all involved in the transduction of mitogenic signals. The findings have lead to the following conclusions: All mitogens but one stimulated Raf-1 kinase activity. Raf- 1 activation was accompanied b y increased Raf- 1 phosphorylation, leading to a characteristic shift in apparent molecular weight (App et al., 1991; Blackshear et al., 1990; Carroll et al., 1990; K. Dell and L. T. Williams, unpublished observations; Kovacina et al., 1990; Morrison et al., 1988, 1989; Siege1 et al., 1990; Thompson et al., 1991; B. C. Turner et al., 1991). Depending on the receptor system, the entire pool of cellular Raf- 1 is activated within 1-20 min and returns to ground levels within 30-120 min in the absence of stimulation. The only mitogen that so far has failed to activate Raf-1 kinase is interleukin-4 (IL-4). Stimulation with I L 4 also resulted in only intermediate growth induction, indicating that I L 4 is not a complete mitogen (B. C. Turner et al., 1991).
1,2: ras regulated?
3: PKC regulated? FIG. 1. Growth factor receptor activation of Raf-1. These receptor systems include receptors with intrinsic or with associated PTK. Ligand-dependent Raf- 1 kinase activation can be triggered by both receptor categories. Several independent pathways exist for Raf coupling: tyrosine phosphorylation, PKC-dependent serine phosphorylation, and PKCindependent serine phosphorylation. Receptors with intrinsic PTK couple predominantly via the latter pathway; receptors with associated P T K couple predominantly via tyrosine phosphorylation, the exception being TCR and Thy-I in 2B4 cells, which exclusively couple via PKC-dependent serine phosphorylation. Ras function is presumably required for stoichiometric Raf-1 activation since blocking YUS. by use of an inducible dominant negative YUS mutant construct inhibits Raf-1 shift induction by serum (U. R. Rapp and G. M. Cooper, unpublished). Ras control of Raf-1 activation may occur at the level of receptor complex formation, Raf- 1 phosphorylation, or may involve regulation of Raf- 1activating second messengers.
56
GISELA HEIDECKER ET AL.
A. MODESOF Raf-1 ACTIVATION The type of Raf-1 phosphorylation that was detected early after receptor engagement varied, depending on the receptor system as well as the cell type in which the activation was analyzed. Basically, four different modes of Raf-1 activation have been observed so far (Table I and Fig. 1): 1. Activation concomitant with protein kinase C-independent phosphorylation on serine residues. 2. Activation following phosphorylation mostly on serines and to a limited extent (less than 1% of the Raf-1 molecules) on tyrosines. 3. Activation accompanied by phosphorylation on tyrosines and serines to about equal levels. 4. Activation with protein kinase C-dependent serine phosphorylation. 1. Protein Kinase C-Independent Sen'nelThreonine Phosphorylation The first mode of activation is most commonly observed following activation of growth factor receptors with intrinsic tyrosine kinase activity. Members of the four structural classes of transmembrane PTK receptors have been analyzed. Ligand binding of insulin receptor (Kovacina et al., 1990; Blackshear et al., 1990) in epithelial cells, and of epidermal growth factor (EGF) receptor (App et al., 1991)and fibroblast growth factor (FGF) receptor (K. Dell and L. T. Williams, unpublished observations) in fibroblasts, was shown to result in an increase of Raf-1 kinase activity, and the same was observed in myeloid cells activated through the colony-stimulating factor (CSF) receptor (Baccarini et al., 1990). In the cases of the EGF and insulin receptors, pretreatment with phorbol ester for 16 hr did not inhibit Raf activation, demonstrating that protein kinase C (PKC) is not responsible for the serine phosphorylation. Indeed, a Raf kinase kinase has been isolated from insulinstimulated cells (Lee et al., 1991). Raf activation following insulin stimulation was also shown to be insensitive to phenyl arsenite oxide, an inhibitor of tyrosine phosphatase that had been used to uncover cryptic tyrosine phosphorylation in other cases (Blackshear et al., 1990), and the increased activity following the stimulation was not diminished by tyrosine-specific phosphatases but could be reduced to basal levels by serine-specific phosphatases (Kovacina et al., 1990). These results taken together suggest that even a minute level of initial tyrosine phosphorylation plays no role in Raf activation in the case of insulin-dependent activation in epithelial cells.
TABLE I MODES OF Raf-ACTIVATION
Cell type
Signal
~
Fibroblast
HeLa-1R cells H5 hepatoma T cells CTLL-2 CTLL-2 CD4 + 2B4 Myeloid cells FDC-PI DA-3 BAC1.2F5 PC12 a
Raf- 1 phosphor y lation on
Kinase activation
Reference
~~
Insulin Insulin
Ser (Tyr)" Ser Ser Ser Ser, Tyr Not determined Ser Ser
IL2 IL-4 Anti-CD4 AntLCD3
Ser, Tyr N o t determined Ser (Tyr)" Ser
IL-3 GMC-SF 1L3 GM-CSF CSF NGF
Ser, Tyr Ser, Tyr Ser (Tyr)" Ser (Tyr)a Ser Ser
PDGF EGF FGF TPA v-src V f i
Morrison et al. (1988, 1989) Morrison et al. (1988); App et al. (1991) Morrison et al. (1988) Morrison et al. (1988) Morrison et al. (1988) Morrison et al. (1988) Kovacina et al. (1990) Blackshear et al. (1990)
+ + +
Only a small fraction of Raf-1 (2%or less) was phosphorylated on tyrosine residues. K. Wood, S. Halegoua, and T. Roberts (personal communication).
B. C. Turner et al. (1991) B. C. Turner et al. (1991) Thompson et al. (1991) Siege1 et al. (1990) Caroll et al. (1990) Caroll et al. ( 1990) Kanakura et al. (1991) Kanakura et al. (1991) Baccarini et al. (1990) In Morrison (1991)b
58
GISELA HEIDECKER E T AL.
2. SerinelThreonine and Low-Level Tyrosine Phosphorylation The only transmembrane PTK growth factor receptor that has been shown to phosphorylate Raf- 1 on tyrosines is platelet-derived growth factor (PDGF) receptor (Morrison et al., 1989). Direct tyrosine phosphorylation was demonstrated when Raf- 1 and PDGF receptor were coproduced in the baculovirus expression system. However, in mouse BALB 3T3 cells, the stoichiometry of the reaction was very low; less than 1% of the Raf molecules were recognized by antiphosphotyrosine antibodies. Indeed, it is possible that not PDGF receptor but an associated tyrosine kinase is responsible for Raf tyrosine phosphorylation, based on the demonstration that several intracellular src family PTKs bind and are activated by PDGF receptor (Kypta et al., 1990). Phosphorylation of Raf- 1 by a transmembrane receptor complex with associated tyrosine kinase has been demonstrated in a human T cell line following CD4 cross-linking. The CD4 transmembrane receptor is bound to the src family tyrosine kinase Lck (Turner et al., 1990; Shaw et al., 1990), which probably directly phosphorylated a small fraction (
tABL- Basic Research Program, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland 21702
I. The raf Oncogene Family Raf- 1 Activation following Growth Factor Stimulation A. Modes of Raf-1 Activation B. Raf-1 Activation in Cells Expressing Oncogenically Activated PTKs 111. Sites of Raf- 1 Phosphorylation A. Sites of Serine/Threonine Phosphorylation B. Sites of Tyrosine Phosphorylation I v. Consequences of Raf-1 Activation A. Raf- 1 in Activated Receptor Complexes B. Relative Position of Raf in the Signaling Cascade C. Substrates of Activated Raf-1 V. Conclusion References 11.
1. The raf Oncogene Family Raf proteins are serine/threonine-specific protein kinases that function in signal transduction, transmitting mitogenic signals from the ligand-activated growth factor receptors at the cell surface to the transcriptional machinery in the nucleus. The general properties of raffamily genes and their role in carcinogenesis have recently been reviewed (Rapp, 1991; Kolch et al., 1990a; Storm et al., 1990a). Briefly, so far three active genes, c-raf-1, A-raf-1, and B-raf have been described in human, mouse, rat, and chicken cells. In mouse and chicken cells (in the latter, the gene is called mil), truncated versions of c-raf-1 and B-raf have been found as oncogenes of acutely transforming viruses (Rapp et al., 1983a,b; Jansen et al., 1983, 1984; Sutrave et al., 1984; Evchene et al., 1990), and all three genes can be oncogenically activated in vitro (Huleihe1 et al., 1986; Heidecker et al., 1990; Sithanandam et al., 1990). They have been mapped to three different chromosomes and are located at Current address: W e c k e AG, Biological Research and Biotechnology, Mooswaldalleel-9, 7800 Freiburg, Germany.
53 ADVANCES IN CANCER RESEARCH, VOL. 58
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
54
GISELA HEIDECKER ET AL.
sites that are frequently altered in human tumors (Sithanandam et al., 1989; Storm et al., 1990a,b). c-ruf-1 is ubiquitously expressed although at variable levels. A-ruf transcripts are most prominent in urogenital tissues and B-raf expression is most abundant in testis and cerebrum (Storm et al., 1990b). T h e raf genes, like most oncogenes, are highly conserved, and homologues have been identified in Xenopur (Le Guellec et al., 1988) and Drosophila (Mark and Rapp, 1984; Mark et al., 1987), in which one of the two gene family members was first identified as the developmental l(1) polehole gene (Ambrosio et al., 1989) acting in concert with the torso gene, which ,encodes a transmembrane phosphotyrosine kinase. A raf-related gene has recently been isolated from CaenorhabditG elegans (Georgi et al., 1990), which combines a Raf-like kinase domain with an amino-terminal region with similarity to the transmembrane and receptor part of transmembrane receptor kinases found in higher organisms. Based on phylogenetic studies, the raf genes, together with mos, encode the only serine/threonine kinases that belong to the src superfamily, which otherwise encodes protein tyrosine kinases (PTKs) (Hanks et al., 1988). 11. Raf-1 Activation following Growth Factor Stimulation
In this article we will concentrate on the evidence for entry of cytosolic Raf- 1 kinase into activated receptor complexes as well as complexdependent or -independent phosphorylation of Raf- 1 on tyrosine and serine residues, which leads to enzyme activation. So far, very little work in this area has addressed the roles of A- o r B-raJ T h e involvement of Raf-1 has been analyzed in more than a dozen receptor systems, all involved in the transduction of mitogenic signals. The findings have lead to the following conclusions: All mitogens but one stimulated Raf-1 kinase activity. Raf- 1 activation was accompanied b y increased Raf- 1 phosphorylation, leading to a characteristic shift in apparent molecular weight (App et al., 1991; Blackshear et al., 1990; Carroll et al., 1990; K. Dell and L. T. Williams, unpublished observations; Kovacina et al., 1990; Morrison et al., 1988, 1989; Siege1 et al., 1990; Thompson et al., 1991; B. C. Turner et al., 1991). Depending on the receptor system, the entire pool of cellular Raf- 1 is activated within 1-20 min and returns to ground levels within 30-120 min in the absence of stimulation. The only mitogen that so far has failed to activate Raf-1 kinase is interleukin-4 (IL-4). Stimulation with I L 4 also resulted in only intermediate growth induction, indicating that I L 4 is not a complete mitogen (B. C. Turner et al., 1991).
1,2: ras regulated?
3: PKC regulated? FIG. 1. Growth factor receptor activation of Raf-1. These receptor systems include receptors with intrinsic or with associated PTK. Ligand-dependent Raf- 1 kinase activation can be triggered by both receptor categories. Several independent pathways exist for Raf coupling: tyrosine phosphorylation, PKC-dependent serine phosphorylation, and PKCindependent serine phosphorylation. Receptors with intrinsic PTK couple predominantly via the latter pathway; receptors with associated P T K couple predominantly via tyrosine phosphorylation, the exception being TCR and Thy-I in 2B4 cells, which exclusively couple via PKC-dependent serine phosphorylation. Ras function is presumably required for stoichiometric Raf-1 activation since blocking YUS. by use of an inducible dominant negative YUS mutant construct inhibits Raf-1 shift induction by serum (U. R. Rapp and G. M. Cooper, unpublished). Ras control of Raf-1 activation may occur at the level of receptor complex formation, Raf- 1 phosphorylation, or may involve regulation of Raf- 1activating second messengers.
56
GISELA HEIDECKER ET AL.
A. MODESOF Raf-1 ACTIVATION The type of Raf-1 phosphorylation that was detected early after receptor engagement varied, depending on the receptor system as well as the cell type in which the activation was analyzed. Basically, four different modes of Raf-1 activation have been observed so far (Table I and Fig. 1): 1. Activation concomitant with protein kinase C-independent phosphorylation on serine residues. 2. Activation following phosphorylation mostly on serines and to a limited extent (less than 1% of the Raf-1 molecules) on tyrosines. 3. Activation accompanied by phosphorylation on tyrosines and serines to about equal levels. 4. Activation with protein kinase C-dependent serine phosphorylation. 1. Protein Kinase C-Independent Sen'nelThreonine Phosphorylation The first mode of activation is most commonly observed following activation of growth factor receptors with intrinsic tyrosine kinase activity. Members of the four structural classes of transmembrane PTK receptors have been analyzed. Ligand binding of insulin receptor (Kovacina et al., 1990; Blackshear et al., 1990) in epithelial cells, and of epidermal growth factor (EGF) receptor (App et al., 1991)and fibroblast growth factor (FGF) receptor (K. Dell and L. T. Williams, unpublished observations) in fibroblasts, was shown to result in an increase of Raf-1 kinase activity, and the same was observed in myeloid cells activated through the colony-stimulating factor (CSF) receptor (Baccarini et al., 1990). In the cases of the EGF and insulin receptors, pretreatment with phorbol ester for 16 hr did not inhibit Raf activation, demonstrating that protein kinase C (PKC) is not responsible for the serine phosphorylation. Indeed, a Raf kinase kinase has been isolated from insulinstimulated cells (Lee et al., 1991). Raf activation following insulin stimulation was also shown to be insensitive to phenyl arsenite oxide, an inhibitor of tyrosine phosphatase that had been used to uncover cryptic tyrosine phosphorylation in other cases (Blackshear et al., 1990), and the increased activity following the stimulation was not diminished by tyrosine-specific phosphatases but could be reduced to basal levels by serine-specific phosphatases (Kovacina et al., 1990). These results taken together suggest that even a minute level of initial tyrosine phosphorylation plays no role in Raf activation in the case of insulin-dependent activation in epithelial cells.
TABLE I MODES OF Raf-ACTIVATION
Cell type
Signal
~
Fibroblast
HeLa-1R cells H5 hepatoma T cells CTLL-2 CTLL-2 CD4 + 2B4 Myeloid cells FDC-PI DA-3 BAC1.2F5 PC12 a
Raf- 1 phosphor y lation on
Kinase activation
Reference
~~
Insulin Insulin
Ser (Tyr)" Ser Ser Ser Ser, Tyr Not determined Ser Ser
IL2 IL-4 Anti-CD4 AntLCD3
Ser, Tyr N o t determined Ser (Tyr)" Ser
IL-3 GMC-SF 1L3 GM-CSF CSF NGF
Ser, Tyr Ser, Tyr Ser (Tyr)" Ser (Tyr)a Ser Ser
PDGF EGF FGF TPA v-src V f i
Morrison et al. (1988, 1989) Morrison et al. (1988); App et al. (1991) Morrison et al. (1988) Morrison et al. (1988) Morrison et al. (1988) Morrison et al. (1988) Kovacina et al. (1990) Blackshear et al. (1990)
+ + +
Only a small fraction of Raf-1 (2%or less) was phosphorylated on tyrosine residues. K. Wood, S. Halegoua, and T. Roberts (personal communication).
B. C. Turner et al. (1991) B. C. Turner et al. (1991) Thompson et al. (1991) Siege1 et al. (1990) Caroll et al. (1990) Caroll et al. ( 1990) Kanakura et al. (1991) Kanakura et al. (1991) Baccarini et al. (1990) In Morrison (1991)b
58
GISELA HEIDECKER E T AL.
2. SerinelThreonine and Low-Level Tyrosine Phosphorylation The only transmembrane PTK growth factor receptor that has been shown to phosphorylate Raf- 1 on tyrosines is platelet-derived growth factor (PDGF) receptor (Morrison et al., 1989). Direct tyrosine phosphorylation was demonstrated when Raf- 1 and PDGF receptor were coproduced in the baculovirus expression system. However, in mouse BALB 3T3 cells, the stoichiometry of the reaction was very low; less than 1% of the Raf molecules were recognized by antiphosphotyrosine antibodies. Indeed, it is possible that not PDGF receptor but an associated tyrosine kinase is responsible for Raf tyrosine phosphorylation, based on the demonstration that several intracellular src family PTKs bind and are activated by PDGF receptor (Kypta et al., 1990). Phosphorylation of Raf- 1 by a transmembrane receptor complex with associated tyrosine kinase has been demonstrated in a human T cell line following CD4 cross-linking. The CD4 transmembrane receptor is bound to the src family tyrosine kinase Lck (Turner et al., 1990; Shaw et al., 1990), which probably directly phosphorylated a small fraction (
