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CNK1 Is a Scaffold Protein That Regulates Src-mediated Raf-1 Activation*

Raf-1 is a regulator of cellular proliferation, differentiation, and apoptosis. Activation of the Raf-1 kinase activity is tightly regulated and involves targeting to the membrane by Ras and phosphorylation by various kinases, including the tyrosine kinase Src. Here we demonstrate that the connector enhancer of Ksr1, CNK1, mediates Src-dependent tyrosine phosphorylation and activation of Raf-1. CNK1 binds preactivated Raf-1 and activated Src and forms a trimeric complex. CNK1 regulates the activation of Raf-1 by Src in a concentration-dependent manner typical for a scaffold protein. Down-regulation of endogenously expressed CNK1 by small inhibitory RNA interferes with Src-dependent activation of ERK. Thus, CNK1 allows cross-talk between Src and Raf-1 and is essential for the full activation of Raf-1.

Raf-1 is a regulator of cellular proliferation, differentiation, and apoptosis. Activation of the Raf-1 kinase activity is tightly regulated and involves targeting to the membrane by Ras and phosphorylation by various kinases, including the tyrosine kinase Src. Here we demonstrate that the connector enhancer of Ksr1, CNK1, mediates Src-dependent tyrosine phosphorylation and activation of Raf-1. CNK1 binds preactivated Raf-1 and activated Src and forms a trimeric complex. CNK1 regulates the activation of Raf-1 by Src in a concentrationdependent manner typical for a scaffold protein. Downregulation of endogenously expressed CNK1 by small inhibitory RNA interferes with Src-dependent activation of ERK. Thus, CNK1 allows cross-talk between Src and Raf-1 and is essential for the full activation of Raf-1.
The serine/threonine kinase Raf plays a key role in many different signaling cascades by transmitting proliferative, developmental, and anti-apoptotic signals from the plasma membrane to the nucleus (1)(2)(3). Vertebrate cells express three different Raf proteins, Raf-1, A-Raf, and B-Raf (Fig. 1A). In contrast to vertebrates, flies and worms express only one of the Raf isoforms corresponding to B-Raf. Genetic and biochemical studies have established that Raf kinases couple the small GTPase Ras to the mitogen-activated protein (MAP) 1 kinase cascade (4). This cascade is conserved throughout evolution and consists of Raf, the dual specificity kinase MEK, and the MAP kinase ERK. Downstream of this cascade, activated ERK phosphorylates a number of cytoplasmic and nuclear targets (2,5).
Activation of Raf is a complex process involving proteinprotein interactions, lipid interactions, and phosphorylation (6,7). Upon mitogenic stimulation, Raf is recruited to the plasma membrane by Ras (8,9). In addition, phosphorylation of Raf is a critical step for Raf activation (10). In the case of Raf-1, negative regulation of the kinase activity involves phosphorylation of Ser-259 by Akt/protein kinase B (11) and of Ser-43 and Ser-233 by protein kinase A (12,13). Full activation of Raf-1 requires the dephosphorylation of Ser-259 and the phosphoryl-ation of several critical residues at the N-terminal side of the catalytic domain, including the phosphorylation of Ser-338 by p21-activated kinase (PAK) or by uncharacterized Ras-activated kinases and the phosphorylation of Tyr-341 by the nonreceptor tyrosine kinase Src (2, 14 -16). In addition, the phosphorylation of Raf-1 at Thr-491 and Ser-494 located in the kinase activation loop is necessary for Raf-1 activation (17). In the case of B-Raf there are two aspartic residues at the position equivalent to Tyr-340 and Tyr-341 in Raf-1, which were proposed to mimic Src-dependent phosphorylation (4,15). In addition, the residue corresponding to the PAK-1 phosphorylation site of Raf-1 is constitutively phosphorylated in the case of B-Raf. These modifications lead to an elevated basal kinase activity of B-Raf compared with Raf-1. The simplified regulation of B-Raf activity may be the reason why a single point mutation can transfer B-Raf in a potent oncoprotein, as was shown recently in human melanomas (18,19).
Recent studies have established that Raf is part of a large multiprotein complex. This complex includes highly abundant proteins such as 14-3-3 as well as heat shock proteins (20,21). Apart from these proteins, genetic studies in yeast and invertebrates revealed the important role of scaffold proteins as organizing centers for signal transduction (22). Mammalian scaffold proteins include the positive regulators of the Raf/ MEK/ERK pathway MP1 (MEK partner 1) and Ksr (kinase suppressor of Ras) (23,24).
In flies, genetic screens for mutations that modify a ksr-dependent phenotype identified a novel gene, the connector enhancer of ksr (cnk). CNK is a multidomain protein and acts as regulator of Ras signaling (25) (Fig. 1A). The N-terminal portion of CNK strongly cooperates with RasV12G37, a Ras effector loop mutant known to activate the Ral pathway in mammalian cells. The C-terminal portion of CNK binds to D-Raf and blocks Ras-and Raf-dependent signaling, probably by titrating out specific signal molecules (26,27). Thus, Drosophila CNK can act on several signaling pathways downstream of Ras and upstream or in parallel to Raf. The importance of CNK in the Ras cascade has been shown in insect cells by a downregulation of CNK that correlates with the reduction of ERKdependent insulin stimulation (28).
Here we report on the direct interaction between human CNK1 and Raf-1. We demonstrate that CNK1 interacts with preactivated forms of Raf-1. In addition, CNK1 interacts with Src and is phosphorylated on tyrosine residue(s) via the Src kinase. CNK1 regulates the Src-mediated Raf-1 activation in the concentration-dependent manner typical for scaffold proteins. Knock-down of CNK1 by small inhibitory RNA (siRNA) resulted in reduction of vascular endothelial growth factor (VEGF)-dependent ERK stimulation, a pathway known to involve Src as an activator of Raf-1 (36). Thus, CNK1 allows a cross-talk between Src and Raf-1, which is essential for full activation of Raf-1.

MATERIALS AND METHODS
Plasmids-The cDNA of human CNK1 was subcloned from pBS-CNK1 (provided by M. Therrien and G. M. Rubin) into pcDNA3 (Invitrogen). The coding sequences for the HA and FLAG tags were inserted 5Ј to the start codon. CNKnt contains the sequence 5Ј to the Aat II restriction site (nucleotide position 939), and CNKct contains the sequence 3Ј to the Aat II site. Wild-type Src cDNA was purchased from Upstate Biotechnology. The Raf1, MEK, and Ras constructs were described previously (37,38).
Cell Culture, Transfection, and Retroviral Transduction-Human embryonic kidney 293T (HEK293T) cells and human epithelial HEp2 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. Transfection of HEK293T and HEp2 cells were performed by the calcium-phosphate precipitation method (40) or the Lipofectamine 2000 method (Invitrogen). For starvation of cells, the medium was replaced by Dulbecco's modified Eagle's medium containing 0.1% fetal calf serum 24 h after transfection, and cells were incubated for an additional 18 h. For inhibition of Src kinase activity, cells were preincubated with the Src inhibitor PP2 (Calbiochem Inc.) 1 h before cell lysis. Stimulation of cells with VEGF (Sigma) were performed as indicated.
Recombinant retrovirus was generated by cotransfection of HEK293T cells with the retroviral vector pMSCV⌬3ЈLTR encoding the siRNA and the two packaging plasmids pVPack-GP and pVPack VSV-G (Stratagene). The virus-containing supernatant was filtered through a 0.45-m filter and used for transduction of HEp2 cells. Stably transduced cells were selected with medium containing 2 g/ml puromycin.
Immunoprecipitation-Cells were lysed in NETN buffer (0.5% Nonidet P-40, 20 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 1 mM EDTA supplemented with 50 mM NaF, 1 mM Na 3 VO 4 , 1 mM phenylmethylsulfonyl fluoride, and 10 g/ml leupeptin) for 20 min at 4°C. Cell debris was pelleted, and the lysate was precleared with 20 l of protein G-Sepharose (Amersham Biosciences). The supernatant was incubated for 3 h at 4°C with an appropriate antibody pre-coupled to protein G-Sepharose. Immunocomplexes were collected by centrifugation, washed five times in lysis buffer, and separated by SDS-PAGE. The proteins were analyzed by immunoblotting using the ECL detection kit (Amersham Biosciences).
For analysis of the trimeric complex, lysates containing FLAG-Raf, HA-CNK, and Src were first immunoprecipitated with an anti-FLAG antibody. The immunoprecipitates were washed extensively, and FLAG-tagged proteins were eluted three times for 15 min each time with anti-FLAG peptide (Sigma) in NETN buffer. One-twentieth of the eluate was used for direct analysis of bound proteins, and the residual eluate was immunoprecipitated with the anti-HA antibody. Samples were separated by electrophoresis with 8% SDS-polyacrylamide gels and analyzed by immunoblotting.

CNK1 Interacts with Preactivated Raf-1-Recent genetic studies in
Drosophila identified the protein CNK as a novel molecule required for signaling via Ras effector proteins (25).
Here we analyzed the role of human CNK1 in the Raf-1 signaling pathway.
First, we tested whether Raf-1 can directly interact with CNK1. HEK293T cells were cotransfected with plasmids encoding HA-tagged CNK and different FLAG-tagged Raf constructs (see Fig. 1A). Immunoprecipitates of Raf proteins were probed with the anti-HA antibody to demonstrate binding of CNK to Raf (Fig. 1B, top). The interaction between the wildtype proteins of Raf and CNK was detectable but weak. Coexpression of activated RasV12 enhanced the association between the two proteins, indicating that Raf-1 preactivated by RasV12 undergoes a conformational change that improves its binding to CNK1. The N terminus of Raf (Rafnt) and the C terminus of Raf (Rafct) bound equally well to CNK, indicating that there are two binding sites for CNK on Raf-1. Coexpression of RasV12 did not affect either one of these interactions. To mimic the activation of Raf-1, we analyzed the constitutively active Raf mutants Raf-S259A and Raf-Y340D (41) (Fig. 1B, bottom  section). These mutants interacted with CNK without the help of RasV12. A kinase-defective Raf mutant, Raf-K375E, and a mutant defective in binding to activated Ras, Raf-R89L, showed only residual binding to CNK1 in unstimulated cells. In RasV12-activated cells, both Raf mutants were able to bind to CNK1 (Fig. 1B, bottom section).
Because B-Raf, the human homologue of Drosophila D-Raf, has an elevated basal kinase activity, we include this Raf protein in our study. In contrast to Raf-1, B-Raf bound to CNK1 without the coexpression of activated Ras (Fig. 1C). The interaction between CNK1 and B-Raf was preferentially exerted via the C terminus of CNK1 (Fig. 1C).
To support the biological relevance of the interaction between CNK1 and Raf-1, we analyzed endogenously expressed proteins. Expression of CNK1 was well detectable in different epithelial cell lines such as HEp2, MCF10A, and HeLa, and lower levels were present in HEK293 cells. Coprecipitation of endogenously expressed CNK1 and Raf-1 was well detectable in epithelial cells, but only after the coexpression of activated Ras (Fig. 1D). Replacing RasV12-dependent stimulation of cells by mitogens did not result in a detectable association between Raf-1 and CNK1 (data not shown), perhaps due to the activation of only a small subpopulation of the cytosolic pool of Raf-1 by mitogens (9). These data show that the interaction between CNK1 and Raf-1 depends on the activation state of the cells.
Dimerization of CNK1 Is Induced by Activation of the MAP Kinase Pathway-Genetic and biochemical data showed that scaffold proteins like Ste5 in yeast or the Jun-interacting protein JIP in mammals can form dimers or even oligomers, which are important for exerting their biological function (42,43). To analyze whether the multidomain protein CNK1 can form dimers or oligomers, two differentially tagged CNK proteins, HA-CNKwt and FLAG-CNKwt, were coexpressed in HEK293T cells. This assay might lead to an underestimation of complex formation, because FLAG-CNK/HA-CNK heterodimers, but not homodimers, can be identified under these conditions. In the first set of the experiment the dimerization of CNK proteins was only weakly detectable. However, coexpression of activators of the MAP kinase pathway such as RasV12 or the constitutively active MEK mutant MEK-Glu-217/Glu-221 (MEK-EE in Fig. 2) strongly induced dimerization of the CNK proteins (Fig. 2). CNK1 is phosphorylated through the MEK pathway, which may trigger CNK1 dimerization. 2 MEK-dependent phosphorylation has been shown also for CNK2 (33). Transient activation of the MAP kinase pathway by treatment of cells with epidermal growth factor or phorbol 12-myristate 13-acetate did not induce dimerization of CNK proteins (data not shown). This result indicates that strong or prolonged activa-2 A. Ziogas, K. Moelling, and G. Radziwill, unpublished observation. tion of the MAP kinase pathway is necessary to trigger formation of CNK dimers or oligomers.
CNK1 Interacts with Src-The primary structure of CNK1 predicts a multidomain protein without a catalytic function, suggesting a function as a scaffold protein, e.g. for the MAP kinase pathway. To test this possibility, we searched for interacting partners of CNK1. Whereas the downstream effectors of Raf-1, MEK, and ERK did not bind, the upstream activator of Raf-1, Src, associated with HA-CNKwt (Fig. 3, top section). This interaction depended on the N-terminal part of CNK, because CNKwt and CNKnt, but not CNKct, retained interaction with Src. Src also induced phosphorylation of CNK1 on tyrosine residues, but, in contrast to binding, phosphorylation of CNK1 took place at its C terminus (Fig. 3, second section from top). A kinase-defective Src mutant, used as control, did not lead to tyrosine phosphorylation of CNK1 (data not shown). These results identified Src as a novel binding partner for CNK1, which could act thereby as an additional scaffold in Raf signaling.
CNK1 Simultaneously Binds to Raf-1 and Src-The maximal activation of Raf-1 requires cooperation of the Ras-dependent recruitment to the plasma membrane and the Src-dependent phosphorylation on tyrosine residues. Our interaction studies indicate that CNK might function as a scaffold protein for Raf-1 and Src. Therefore, we tested to determine whether CNK1 could bind Raf-1 and Src simultaneously in a trimeric complex. FLAG-Rafwt was coexpressed with Src-wt in the presence or absence of HA-tagged CNKwt. Raf was immunoprecipitated with the anti-FLAG antibody, and the immunoprecipitates were eluted from the antibody with the FLAG peptide (Fig. 4,  top section). Eluates, which contained equal amounts of Raf, were immunoprecipitated with the anti-HA antibody and immunoblotted with the anti-Src antibody. This sequential precipitation showed that Rafwt indeed formed a trimeric complex with CNK and Src (Fig. 4, second section from top). A kinasedefective mutant of Raf-1, Raf K375E, which only has a residual affinity to CNK (Fig. 1B), did not form a complex with CNK and Src. These results show that CNK1, Src, and Raf-1 form a trimeric complex and strongly support the notion that CNK1 may act as a scaffold to regulate Raf-1 activation.
CNK1 Regulates Src-mediated Raf-1 Activation-One function of scaffold proteins is to organize and thereby regulate signaling pathways. Therefore, we analyzed the effect of CNK1 on Raf-1 activation and Raf-1-dependent stimulation of MEK and ERK. Ectopic expression of CNK1 had no effect on the basal kinase activity of Raf-1, MEK, or ERK (data not shown). However, CNK1 interfered with the Src-dependent activation of Raf-1.
Constant amounts of Src and FLAG-Rafwt were coexpressed with an increasing amount of HA-CNKwt in HEK293T cells (Fig. 5A). Without coexpression of HA-CNKwt, Src induced a weak tyrosine phosphorylation of Raf. Coexpression of a low amount of HA-CNKwt increased tyrosine phosphorylation of Raf-1. Higher levels of HA-CNKwt led to the opposite effect, the loss of Src-dependent Raf-1 phosphorylation. ERK phosphorylation behaved similarly to Raf-1 tyrosine phosphorylation. Low levels of CNKwt increased ERK phosphorylation, whereas higher levels of CNKwt decreased it.
Next, we tested to determine which domain of CNK1 is required for Src-dependent Raf activation. In this case, the activation of Raf-1 was monitored by in vitro kinase assays using immunopurified Raf as the kinase and MEK fused to glutathione S-transferase (GST-MEK) as a Raf-specific substrate. Consistent with tyrosine phosphorylation of Raf-1 (Fig.  5A), low levels of the wild-type CNK1 protein increased Src-dependent Raf activation, whereas higher levels decreased it (Fig.   FIG. 2. CNK  5B). In addition, large amounts of CNKnt, but not CNKct, significantly decreased Src-dependent Raf activation (compare 0.5 g and 2.5 g of DNA), probably because CNKnt, but not CNKct, titrate out Src, which binds just to the N-terminal part of CNK1 (Fig. 3).
These results indicate that CNK1 regulates the Src-dependent activation of Raf-1 only under optimized conditions. Too low or too high amounts of CNK1 prevented the formation of the trimeric complex and promoted dimerization between CNK1 and Src or Raf-1, thereby preventing activation of Raf-1 by Src.
Down-regulation of CNK1 Impairs VEGF-dependent ERK Phosphorylation-We showed that CNK1 transmits the signal from active Src to the Raf pathway. The growth factor VEGF specifically induces tyrosine phosphorylation and activation of Raf-1 via Src (36). This pathway results in ERK stimulation and protects cells from apoptosis. Because CNK1 is well expressed in epithelial cells and because the expression of VEGF receptors is not restricted to endothelial cells (44,45), we have chosen epithelial cells to analyze a putative function of CNK1 in VEGF-dependent signaling.
To knock-down CNK1 in HEp2 cells, we stably expressed siRNA specifically targeting CNK1 (Fig. 6, top section). For the control, siRNA against firefly luciferase (GL2) was used. VEGF stimulated ERK phosphorylation in control cells, but this stimulation was impaired in CNK1-depleted cells (Fig. 6, second section from the top, compare third and fourth lanes from the left). As expected, Src mediated VEGF-dependent stimulation of ERK, because pretreatment of cells with the Src inhibitor PP2 reduced ERK phosphorylation (Fig. 6, second section from  the top, compare third and fifth lanes from the left). As shown in Fig. 5A, tyrosine phosphorylation and activation of Raf-1 by Src is modulated by the amount of the scaffold protein CNK1. In CNK1-depleted cells there is no VEGF-induced tyrosine phosphorylation of endogenous Raf-1 detectable, whereas this is the case in the control cells (Fig. 6, bottom two sections). These data show that CNK is involved in VEGF-dependent stimulation of ERK, a pathway mediated by Src and Raf-1 DISCUSSION Raf-1 activation is a multistep process. It involves recruitment of Raf-1 to the plasma membrane by active Ras-GTP and the phosphorylation of Raf-1 at Ser-338 by Ras-activated kinases (15,16). Full activation of Raf-1 also requires tyrosine FIG. 4. CNK forms a ternary complex with Raf-1 and Src in vivo. HEK293T cells were cotransfected with plasmids (3 g each) coding for HA-CNKwt, FLAG-Rafwt, and Srcwt as indicated. Cells were lysed, and FLAG-Raf was immunopurified with anti-FLAG antibody (top section). Bound proteins were eluted with a FLAG peptide, and eluates were subsequently immunoprecipitated (IP) with an anti-HA antibody and immunoblotted with an anti-Src antibody (second section from top). A small aliquot of cell lysate was used to monitor expression of Src or HA-CNKwt (bottom two sections). FLAG-RafK375E is a kinaseinactive mutant of Raf-1.

FIG. 5. CNK regulates Src-mediated Raf-1 activation.
A, FLAG-Rafwt (1 g), Srcwt (0.5 g), and increasing amounts of HA-CNKwt (0.1, 0.5, and 2.5 g) were expressed in HEK293T cells. Raf proteins were immunoprecipitated (IP) with an anti-FLAG antibody, and tyrosine-phosphorylated activated Raf (pRaf) was monitored by immunoblotting (Blot) with an anti-Tyr(P)-340/Tyr(P)-341 Raf antibody (anti-pRaf YY340/341; rabbit anti-c-Raf (pYpY340/341) from BioSource). Direct cell lysates were subjected to immunoblot analysis with antiphospho-ERK (pERK) and anti-ERK (ERK) to detect activated ERK and the total amount of ERK, respectively. Expressions of CNK, Raf, and Src were controlled by immunoblotting with the appropriate antibodies. B, HEK293T cells were transfected with the constructs as indicated. Immunopurified Raf was analyzed by in vitro kinase assays using GST-MEK as a specific substrate for Raf. The expression of FLAG-Raf (bottom section), HA-CNK, and Src (data not shown) were controlled by immunoblotting. Numbers under the sections indicate fold activation. phosphorylation of Raf-1 at Tyr-341. This is mediated by Src or Src-like kinases (15,46). Activated Raf-1 can transmit the signal to MEK, ERK, and downstream effectors. This process is coordinated by two known scaffold proteins, Ksr and MP-1 (MEK partner 1), which connect MEK and ERK by directly binding to both of them (23,24,47). Here, we describe the multidomain protein CNK1 as a scaffold that connects Src and Raf-1 and thereby allows Src-dependent activation of Raf-1.
We demonstrate that CNK1 binds to preactivated Raf-1. Wildtype Raf-1 bound only weakly to CNK1, whereas Raf-1 activated by Ras bound strongly to CNK1. A preactivated state as a requirement for binding may explain why, in former studies, an interaction between ectopically expressed Raf-1 and CNK1 has not been detected (25). The biological relevance of the interaction between Raf-1 and CNK1 is supported by coimmunoprecipitation of both proteins endogenously expressed in epithelial cells. Also, under these conditions the complex formation between Raf-1 and CNK1 is strengthened by activation of the cells (Fig. 1D).
Raf-1 mutants that have an elevated basal kinase activity also showed an increased binding to CNK1. The mutant Raf-S259A does not bind 14-3-3 proteins at its N terminus and therefore lacks negative regulation of its kinase activity (41). This mutation leads to activation similar to that induced by Ras, which also displaces 14-3-3 from binding to Raf Ser-259. In both cases, a conformational change and pre-activation of Raf-1 is induced, which is a prerequisite for the binding of Raf-1 to CNK1.
The mutant Raf-Y340D, in which one tyrosine residue is substituted by the negatively charged aspartic acid, mimics Src-dependent activation (46). This mutant resembles B-Raf and Drosophila D-Raf, both of which contain two aspartic amino acid residues at the positions equivalent to Tyr-340 and Tyr-341 of Raf-1. These Raf proteins no longer require activa-tion by Src but exhibit a constitutive elevated basal kinase activity. D-Raf has been shown to bind to the Drosophilaspecific CNK in insect cells (25). This finding is in agreement with the result described here demonstrating that B-Raf, the homologue of D-Raf, can bind to mammalian CNK1. This interaction is not further influenced by coexpression of activated Ras, showing once more that pre-activation of Raf allows an efficient interaction with CNK1.
Preactivation of Raf-1 may depend on a conformational change induced by binding to activated Ras or by phosphorylation via Ras-activated kinases. Association of CNK1 with the kinase-defective mutant of Raf, RafK375E, and the mutant RafR89L, impaired for binding to active Ras, can be induced by coexpression of RasV12. This observation indicates that Rasactivated kinases, known to phosphorylate Ser-338 and possibly also Thr-491 and/or Ser-494 in the kinase activation loop of Raf-1, could be responsible for the postulated conformational change (16).
The binding region of Raf to mammalian CNK proteins is localized at the C-terminal part of CNK1 but is not further characterized. In Drosophila, D-CNK interacts via its C-terminal Raf-interacting and Raf inhibitory region (RIR) with D-Raf (26). This sequence is unique to D-CNK. In a very recent paper it has been shown that Drosophila Src42, independently of its catalytic function, binds near RIR and thereby counteracts the inhibitory effect of the RIR on D-Raf (48).
Our studies reveal another mechanism for Src activation of Raf-1 via CNK1 that involves a catalytic active Src. The simultaneous binding of active Src and preactivated Raf-1 to CNK suggests that CNK1 allows Src to phosphorylate and fully activate Raf-1. Thus, CNK1 acts as a scaffold protein that assembles and coordinates two kinases of a single signaling pathway. Typically, scaffold proteins function in a concentrationdependent manner (49,50). Consequently increasing amounts of CNK1 first increase and then decrease Src-dependent activation of Raf-1 (Fig. 5A). A similar behavior has been described for other scaffold proteins such as the MEK partner protein MP-1 and Ksr. MP-1 connects MEK1 and ERK1 downstream of Raf-1 (23). Ksr acts as scaffold that connects MEK and ERK with activated Raf-1 and allows Raf-1 to phosphorylate and activate MEK, which, in turn, activates ERK (47,51). The scaffold protein CNK1 acts even further upstream to activate Raf-1.
The contribution of Ras-and Src-dependent activation of Raf-1 depends on the stimulus and the cell type. It has been well documented for endothelial cells that VEGF signals via Src to Raf/MEK/ERK, whereas the basic fibroblast growth factor (bFGF) mainly involves Ras-and PAK-dependent activation of Raf-1 (36). Both pathways protected cells from apoptosis. The VEGF-and Src-dependent activation of Raf-1 results in phosphorylation of ERK and should involve CNK1 according to the results presented here. Indeed, depletion of CNK1 significantly reduced ERK activation by VEGF, indicating that CNK-dependent steps participate in VEGF-induced signaling. Activation of ERK by the basic fibroblast growth factor was not significantly affected in CNK1-depleted cells (data not shown), suggesting that the Ras-and PAK-dependent activation of the Raf/MEK/ERK pathway is not sensitive to CNK1 under these conditions. This is consistent with a recent observation that CNK1 does not contribute to epithelial growth factor-dependent activation of ERK in epithelial cells (31).
There is growing evidence obtained by knock-out studies that MEK is not the only substrate for Raf-1 activity (52,53). Raf-1 exerts an anti-apoptotic response by phosphorylating and thereby inactivating the pro-apoptotic Bcl-2 family member Bad (54). Recently it has been reported that overexpression of CNK1 can support pro-apoptotic signaling under certain conditions, but it is unclear how endogenous levels of CNK1 will behave (55). Therefore, there could be a link between CNK and Raf in the regulation of apoptosis.
Src-dependent phosphorylation and activation is specific for Raf-1, but the binding of CNK1 to both Raf-1 or to B-Raf suggests additional functions for the CNK-Raf complex. Recently it has been demonstrated that Raf-1 and B-Raf can heterodimerize (56). Furthermore, it was proposed that B-Raf mutants with impaired kinase activity can still activate ERK by stimulating Raf-1 (19). An intriguing possibility would be that CNK1 participates in Raf-1/B-Raf heterodimerization and Raf-1 activation by B-Raf because it interacts with both Raf family members (see Fig. 1).
As is the case for Raf proteins, CNK1 is cytosolic in unstimulated cells and recruited to the plasma membrane upon stimulation. 2 Therefore, an additional function of a CNK1-Raf complex could be to shuttle activated membrane-bound Raf from the site of stimulation to the site of its substrate. Dimerization of CNK1 as shown here may be involved in this process, although this has still to be demonstrated.
Another function of CNK1 could be to allow a concerted action of active Src and fully activated Raf-1 or B-Raf for dual-phosphorylation of downstream targets. A substrate that is phosphorylated at tyrosine residues by Src and on serine residues by the Raf/MEK/ERK pathway is cortactin. Phosphorylation of cortactin is linked to actin organization and cell motility (57). Interestingly, the focal adhesion complex protein paxillin forms a complex with Raf-1, MEK, and ERK to facilitate localized ERK activation (58). Binding and activation of ERK depends on prior tyrosine phosphorylation of paxillin by Src. CNK1 binds to Src and is phosphorylated via Src as shown here (Fig. 3). This tyrosine phosphorylation of CNK1 may create binding sites for substrates of CNK1-associated kinases as described here or for other signaling proteins.
Members of the CNK family are multidomain proteins. Only a few proteins interacting with CNK have been identified. Most of them may be signaling molecules such as GTPases and their effectors and protein kinases as shown here. In addition, CNK2A binds via its C-terminal PDZ domain binding motif to the synaptosomal PDZ proteins PSD-95, S-SCAM (synaptic scaffolding molecule), and densin-180 (29,30). CNK1 also contains a putative PDZ domain binding motif at its very Cterminal end. Therefore, it would be of interest to know whether CNK1 itself, via its PDZ domain-binding motif, may form a complex with other scaffold proteins, which would open up novel aspects for Src and Raf kinases in such a large signal transduction complex.