The kinase activity of kinase suppressor of Ras1 (KSR1) is independent of bound MEK

Kinase Suppressor of Ras1 (KSR1) functions as a positive modulator of Ras-dependent signaling either upstream of or parallel to Raf-1, and pharmacologic inactivation of KSR1 may serve as a treatment for Rasdriven malignancies such as pancreatic cancer (Xing, H. R., Cordon-Cardo, C., Deng, X., Tong, W., Campodonico, L., Fuks, Z., and Kolesnick, R. (2003) Nat. Med. 9, 1266-1268). Although some studies demonstrated a requirement for KSR1 kinase activity for its action, others suggested KSR1 acts primarily as a scaffold facilitating assembly of the c-Raf-1/MEK module. We recently established a two-stage in vitro reconstitution assay to measure KSR1 kinase activity (Xing, H. R., Lozano, J., and Kolesnick, R. (2000) J. Biol. Chem. 275, 17276-17280). In this assay, KSR1, immunopurified to apparent homogeneity, never comes in contact with recombinant kinases other than c-Raf-1. In the first assay stage, activated KSR1 is incubated with recombinant c-Raf-1 and ATP. In the second stage, activated c-Raf-1 is separated from KSR1, and incubated with unactivated MEK1, unactivated MAPK, Elk-1, and ATP. Elk-1 phosphorylation serves as a specific readout for MAPK activation. However, because KSR1 constitutively associates with MEK1 and this interaction appears critical for KSR1 scaffolding function, it has been argued that the kinase activity detected is an artifact of KSR1-bound MEK1. To address these concerns, we depleted as much as 90% of KSR1-bound MEK1 by high salt washing without altering KSR1 kinase activity. Further, a complete inactivation of KSR1-bound MEK1 by pretreating with the MEK inhibitor PD 98059 prior to the first assay stage did not alter KSR1 kinase activity. In addition, the omission of exogenous recombinant GST-MEK1 from the reaction mixture during the second assay stage abolished Elk-1 phosphorylation confirming KSR1-bound MEK1 does not support MAPK activation in our in vitro assay. Moreover, a kinase-inactive mutant, FLAG-Ki-KSR1(D683A/D700A), which efficiently interacts with endogenous MEK1, lacks kinase activity. These results collectively support our contention that the kinase activity of KSR1 is an intrinsic property of this protein independent of KSR1-bound endogenous MEK.     

Kinase suppressor of Ras signals through Thr269 of c-Raf-1

We recently established a two-stage in vitro assay for KSR kinase activity in which KSR never comes in contact with any recombinant kinase other than c-Raf-1 and defined the epidermal growth factor (EGF) as a potent activator of KSR kinase activity (Xing, H. R., Lozano, J., and Kolesnick, R. (2000) J. Biol. Chem. 275, 17276-17280). That study, however, did not address the mechanism of c-Raf-1 stimulation by activated KSR. Here we show that phosphorylation of c-Raf-1 on Thr(269) by KSR is necessary for optimal activation in response to EGF stimulation. In vitro, KSR specifically phosphorylated c-Raf-1 on threonine residues during the first stage of the two-stage kinase assay. Using purified wild-type and mutant c-Raf-1 proteins, we demonstrate that Thr(269) is the major c-Raf-1 site phosphorylated by KSR in vitro and that phosphorylation of this site is essential for c-Raf-1 activation by KSR. KSR acts via transphosphorylation, not by increasing c-Raf-1 autophosphorylation, as kinase-inactive c-Raf-1(K375M) served as an equally effective KSR substrate. In vivo, low physiologic doses of EGF (0.001-0.1 ng/ml) stimulated KSR activation and induced Thr(269) phosphorylation and activation of c-Raf-1. Low dose EGF did not induce serine or tyrosine phosphorylation of c-Raf-1. High dose EGF (10-100 ng/ml) induced no additional Thr(269) phosphorylation, but rather increased c-Raf-1 phosphorylation on serine residues and Tyr(340)/Tyr(341). A Raf-1 mutant with valine substituted for Thr(269) was unresponsive to low dose EGF, but was serine- and Tyr(340)/Tyr(341)-phosphorylated and partially activated at high dose EGF. This study shows that Thr(269) is the major c-Raf-1 site phosphorylated by KSR. Furthermore, phosphorylation of this site is essential for c-Raf-1 activation by KSR in vitro and for optimal c-Raf-1 activation in response to physiologic EGF stimulation in vivo.     

Kinase suppressor of Ras transphosphorylates c-Raf-1

Whether kinase suppressor of Ras1 (KSR1) is an active kinase that phosphorylates c-Raf-1 or a scaffold that coordinates signaling along the Ras/ERK1 signaling module is actively debated. In this study, we generated a monoclonal antibody against a c-Raf-1 peptide containing phosphorylated Thr²⁶⁹, the putative target for KSR1 kinase activity. We show that this antibody detects Thr²⁶⁹-phosphorylated c-Raf-1 in A431 cells upon epidermal growth factor (EGF) stimulation, preceding MEK1 activation. Furthermore, this antibody detects in vitro phosphorylation of FLAG-c-Raf-1 and kinase-dead FLAG-c-Raf-1(K375M) by immunopurified KSR1, but fails to detect phosphorylation of FLAG-c-Raf-1(K375M/T269V), engineered with a Thr²⁶⁹ to valine substitution. To provide unequivocal evidence that KSR1 is a legitimate kinase, we purified KSR1 to homogeneity, confirmed by mass spectrometry, renatured it in-gel, and demonstrated that it phosphorylates BSA-conjugated c-Raf-1 peptide at Thr²⁶⁹. These studies add to emerging data validating KSR1 as a kinase that phosphorylates c-Raf-1.     

Epidermal growth factor (EGF) stimulates association and kinase activity of Raf-1 with the EGF receptor.

Raf-1 serine- and threonine-specific protein kinase is transiently activated in cells expressing the epidermal growth factor (EGF) receptor upon treatment with EGF. The stimulated EGF receptor coimmunoprecipitates with Raf-1 kinase and mediates protein kinase C-independent phosphorylation of Raf-1 on serine residues. Hyperphosphorylated Raf-1 has lower mobility on sodium dodecyl sulfate gels and has sixfold-increased activity in immunocomplex kinase assay with histone H1 or Raf-1 sequence-derived peptide as a substrate. Raf-1 activation requires kinase-active EGF receptor; a point mutant lacking tyrosine kinase activity in inactive in Raf-1 coupling and association. It is noteworthy that tyrosine phosphorylation of c-Raf-1 induced by EGF was not detected in these cells. These observations suggest that Raf-1 kinase may act as an important downstream effector of EGF signal transduction.     

Nm23-H1 metastasis suppressor phosphorylation of kinase suppressor of Ras via a histidine protein kinase pathway

The metastasis-suppressive activity of Nm23-H1 was previously correlated with its in vitro histidine protein kinase activity, but physiological substrates have not been identified. We hypothesized that proteins that interact with histidine kinases throughout evolution may represent partners for Nm23-H1 and focused on the interaction of Arabidopsis "two-component" histidine kinase ERS with CTR1. A mammalian homolog of CTR1 was previously reported to be c-Raf; we now report that CTR1 also exhibits homology to the kinase suppressor of Ras (KSR), a scaffold protein for the mitogen-activated protein kinase (MAPK) cascade. Nm23-H1 co-immunoprecipitated KSR from lysates of transiently transfected 293T cells and at endogenous protein expression levels in MDA-MB-435 breast carcinoma cells. Autophosphorylated recombinant Nm23-H1 phosphorylated KSR in vitro. Phosphoamino acid analysis identified serine as the major target, and two peaks of Nm23-H1 phosphorylation were identified upon high performance liquid chromatography analysis of KSR tryptic peptides. Using site-directed mutagenesis, we found that Nm23-H1 phosphorylated KSR serine 392, a 14-3-3-binding site, as well as serine 434 when serine 392 was mutated. Phosphorylated MAPK but not total MAPK levels were reduced in an nm23-H1 transfectant of MDA-MB-435 cells. The data identify a complex in vitro histidine-to-serine protein kinase pathway, which may contribute to signal transduction and metastasis.     

Phosphorylation of the kinase suppressor of ras by associated kinases

The kinase suppressor of Ras (KSR) is a loss-of-function allele that suppresses the rough eye phenotype of activated Ras in Drosophila and the multivulval phenotype of activated Ras in Caenorhabditis elegans. The physiological role of mammalian KSR is not known. We examined the mechanisms regulating the phosphorylation of this putative kinase in mammalian cells. Wild-type mouse KSR and a mutated KSR protein predicted to create a kinase-dead protein are phosphorylated identically in intact cells and in the immune complex. Phosphopeptide sequencing identified 10 in vivo phosphorylation sites in KSR, all of which reside in the 539 noncatalytic amino terminal amino acids. Expression of the amino terminal portion of KSR alone demonstrated that it was phosphorylated in the intact cell and in an immune complex in a manner indistinguishable from that of intact KSR. These data demonstrate that the kinase domain of KSR is irrelevant to its phosphorylation state and suggest that the phosphorylation of KSR and its association with a distinct set of kinases may affect intracellular signaling.     

Flotillin-1/reggie-2 protein plays dual role in activation of receptor-tyrosine kinase/mitogen-activated protein kinase signaling

Our previous work has shown that the membrane microdomain-associated flotillin proteins are potentially involved in epidermal growth factor (EGF) receptor signaling. Here we show that knockdown of flotillin-1/reggie-2 results in reduced EGF-induced phosphorylation of specific tyrosines in the EGF receptor (EGFR) and in inefficient activation of the downstream mitogen-activated protein (MAP) kinase and Akt signaling. Although flotillin-1 has been implicated in endocytosis, its depletion affects neither the endocytosis nor the ubiquitination of the EGFR. However, EGF-induced clustering of EGFR at the cell surface is altered in cells lacking flotillin-1. Furthermore, we show that flotillins form molecular complexes with EGFR in an EGF/EGFR kinase-independent manner. However, knockdown of flotillin-1 appears to affect the activation of the downstream MAP kinase signaling more directly. We here show that flotillin-1 forms a complex with CRAF, MEK1, ERK, and KSR1 (kinase suppressor of RAS) and that flotillin-1 knockdown leads to a direct inactivation of ERK1/2. Thus, flotillin-1 plays a direct role during both the early phase (activation of the receptor) and late (activation of MAP kinases) phase of growth factor signaling. Our results here unveil a novel role for flotillin-1 as a scaffolding factor in the regulation of classical MAP kinase signaling. Furthermore, our results imply that other receptor-tyrosine kinases may also rely on flotillin-1 upon activation, thus suggesting a general role for flotillin-1 as a novel factor in receptor-tyrosine kinase/MAP kinase signaling.     

A mitogen-activated protein (MAP) kinase activating factor in mammalian mitogen-stimulated cells is homologous to Xenopus M phase MAP kinase activator.

The mitogen-activated protein (MAP) kinases, a family of 40-45-kDa kinases whose activation requires both tyrosine and threonine/serine phosphorylations, are suggested to play key roles in various phosphorylation cascades. A previous study of Krebs and co-workers (Ahn, N. G., Seger, R., Bratlien, R. L., Diltz, C. D., Tonks, N. K., and Krebs, E. G. (1991) J. Biol. Chem. 266, 4220-4227) detected an activity in epidermal growth factor (EGF)-stimulated 3T3 cells that can stimulate inactive MAP kinases. We observed this activity in rat 3Y1 cells treated with various mitogenic factors and in PC12 cells treated with nerve growth factor (NGF). Its kinetics of activation and deactivation following EGF or NGF stimulation roughly paralleled that of MAP kinase. The MAP kinase activator required the presence of ATP and a divalent cation such as Mn2+ and Mg2+ and was inactivated by phosphatase 2A treatment in vitro. This activator has been isolated from EGF-stimulated 3Y1 cells by sequential chromatography and identified as a 45-kDa monomeric protein. It was able to activate mammalian and Xenopus MAP kinases in vitro and was very similar to Xenopus M phase MAP kinase activating factor, which was purified previously from mature oocytes (Matsuda, S., Kosako, H., Takenaka, K., Moriyama, K., Sakai, H., Akiyama, T., Gotoh, Y., and Nishida, E. (1992) EMBO J. 11, 973-982), in terms of its functional, immunological, and physicochemical properties. Thus, the same or a similar upstream activating factor may function in mitogen-induced and M phase-promoting factor-induced MAP kinase activation pathways.     

Activation of c-Raf-1 kinase signal transduction pathway in alpha(7) integrin-deficient mice.

Integrin alpha(7)-deficient mice develop a novel form of muscular dystrophy. Here we report that deficiency of alpha(7) integrin causes an activation of the c-Raf-1/mitogen-activated protein (MAP) 2 kinase signal transduction pathway in muscle cells. The observed activation of c-Raf-1/MAP2 kinases is a specific effect, because the alpha(7) integrin deficiency does not cause unspecific stress as determined by measurement of the Hsp72/73 level and activity of the JNK2 kinase. Because an increased level of activated FAK was found in muscle of alpha(7) integrin-deficient mice, the activation of c-Raf-1 kinase is triggered most likely by an integrin-dependent pathway. In accordance with this, in the integrin alpha(7)-deficient mice, part of the integrin beta(1D) variant in muscle is replaced by the beta(1A) variant, which permits the FAK activation. A recent report describes that integrin activity can be down-modulated by the c-Raf-1/MAP2 kinase pathway. Specific activation of the c-Raf-1/MAP2 kinases by cell-permeable peptides in skeletal muscle of rabbits causes degeneration of muscle fibers. Therefore, we conclude that in alpha(7) integrin-deficient mice, the continuous activation of c-Raf-1 kinase causes a permanent reduction of integrin activity diminishing integrin-dependent cell-matrix interactions and thereby contributing to the development of the dystrophic phenotype.     

KSR1 is a functional protein kinase capable of serine autophosphorylation and direct phosphorylation of MEK1

The extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway is a highly conserved signaling pathway that regulates diverse cellular processes including differentiation, proliferation, and survival. Kinase suppressor of Ras-1 (KSR1) binds each of the three ERK cascade components to facilitate pathway activation. Even though KSR1 contains a C-terminal kinase domain, evidence supporting the catalytic function of KSR1 remains controversial. In this study, we produced recombinant wild-type or kinase-inactive (D683A/D700A) KSR1 proteins in Escherichia coli to test the hypothesis that KSR1 is a functional protein kinase. Recombinant wild-type KSR1, but not recombinant kinase-inactive KSR1, underwent autophosphorylation on serine residue(s), phosphorylated myelin basic protein (MBP) as a generic substrate, and phosphorylated recombinant kinase-inactive MAPK/ERK kinase-1 (MEK1). Furthermore, FLAG immunoprecipitates from KSR1^−/− colon epithelial cells stably expressing FLAG-tagged wild-type KSR1 (+KSR1), but not vector (+vector) or FLAG-tagged kinase-inactive KSR1 (+D683A/D700A), were able to phosphorylate kinase-inactive MEK1. Since TNF activates the ERK pathway in colon epithelial cells, we tested the biological effects of KSR1 in the survival response downstream of TNF. We found that +vector and +D683A/D700A cells underwent apoptosis when treated with TNF, whereas +KSR1 cells were resistant. However, +KSR1 cells were sensitized to TNF-induced cell loss in the absence of MEK kinase activity. These data provide clear evidence that KSR1 is a functional protein kinase, MEK1 is an in vitro substrate of KSR1, and the catalytic activities of both proteins are required for eliciting cell survival responses downstream of TNF.     

Constitutive association of c-N-Ras with c-Raf-1 and protein kinase C epsilon in latent signaling modules

Phorbol ester stimulation of the MAPK cascade is believed to be mediated through the protein kinase C (PKC)-dependent activation of Raf-1. Although several studies suggest that phorbol ester stimulation of MAPK is insensitive to dominant-negative Ras, a requirement for Ras in Raf-1 activation by PKC has been suggested recently. We now demonstrate that in normal, quiescent mouse fibroblasts, endogenous c-N-Ras is constitutively associated with both c-Raf-1 and PKC epsilon in a biochemically silent, but latent, signaling module. Chemical inhibition of novel PKCs blocks phorbol 12-myristate 13-acetate (PMA)-mediated activation of MAPKs. Down-regulation of PKC epsilon protein levels by antisense oligodeoxyribonucleotides blocks MAPK activation in response to PMA stimulation, demonstrating that PKC epsilon activity is required for MAPK activation by PMA. c-Raf-1 activity in immunoprecipitated c-N-Ras.c-Raf-1.PKC epsilon complexes is stimulated by PMA and is inhibited by GF109203X, thereby linking c-Raf-1 activation in this complex to PKC activation. These observations suggest that in quiescent cells Ras is organized into ordered, inactive signaling modules. Furthermore, the regulation of the MAPK cascade by both Ras and PKC is intimately linked, converging at the plasma membrane through their association with c-Raf-1.     

Phosphorylation regulates the nucleocytoplasmic distribution of kinase suppressor of Ras.

KSR (kinase suppressor of Ras) has been proposed as a molecular scaffold regulating the Raf/MEK/ERK kinase cascade. KSR is phosphorylated on multiple phosphorylation sites by associated kinases. To identify potential mechanisms used by KSR to regulate ERK activation, green fluorescent protein was fused to intact and mutated KSR constructs lacking specific phosphorylation sites, and the subcellular distribution of each construct was observed in live cells. Mutation of a subset of KSR phosphorylation sites caused the redistribution of KSR to the nucleus. To determine whether intact KSR is normally imported to the nucleus, REF-52 fibroblasts expressing KSR were treated with 10 nm leptomycin B, which inhibits Crm1-dependent nuclear export. KSR accumulated in the nucleus within 2 h of treatment with leptomycin B, suggesting that KSR cycles continuously through the nucleus. Nuclear import of KSR was blocked by mutations that inhibit the interaction of KSR with MEK. Coexpression of fluorescent forms of KSR and MEK in cells revealed that each protein promoted the localization of the other in the cytoplasm. These data indicate that the subcellular distribution of KSR is dynamically regulated through phosphorylation and MEK interaction in a manner that may affect signaling through ERK.     

Daphnetin, one of coumarin derivatives, is a protein kinase inhibitor.

Protein kinases play key roles in the control of cell proliferation, differentiation and metabolism. In this work, we studied the effect of coumarin and its derivatives, including daphnetin, esculin, 2-OH-coumarin, 4-OH-coumarin and 7-OH-coumarin, on the activity of protein kinases. It was found that, in these compounds, only daphnetin was a protein kinase inhibitor. This compound inhibited tyrosine-specific protein kinase, EGF receptor (IC(50) = 7.67 microM), and serine/threonine-specific protein kinases, including cAMP-dependent protein kinase (PKA) (IC(50) = 9.33 microM) and protein kinase C (PKC) (IC(50) = 25.01 microM) in vitro. The inhibition of EGF receptor tyrosine kinase by daphnetin was competitive to ATP and non-competitive to the peptide substrate. The inhibition of EGF-induced tyrosine phosphorylation of EGF receptor by daphnetin was not observed in human hepatocellular carcinoma HepG2 cells. The structural comparison of daphnetin with coumarin and other coumarin derivatives suggests that the hydroxylation at C8 may be required for daphnetin acting as a protein kinase inhibitor.     

Estrogen modulation of prolactin gene expression requires an intact mitogen-activated protein kinase signal transduction pathway in cultured rat pituitary cells

Expression of the PRL gene is regulated by many factors, including cAMP, estradiol (E2), phorbol esters, epidermal growth factor (EGF), and TRH. The promoter region of the rat PRL gene has been shown to contain DNA sequences that are thought to support the direct interaction of estrogen receptors (ERs) with DNA. It is by this direct ER/DNA interaction that estrogen is thought to modulate expression of PRL. We report here that estrogeninduced PRL expression requires an intact mitogen-activated protein kinase (MAPK) signal transduction pathway in cultured rat pituitary cells (PR1 lactotroph and GH3 somatolactotroph cell lines). Interfering with the MAPK signaling cascade by inhibiting the activity of MAPK kinase (MEK) ablates the ability of estrogen to induce PRL mRNA and protein. In these cell lines, estrogen activates extracellular regulated protein kinases ERK-1 and ERK-2 enzyme activities maximally within 10 min of 1 nM E2 treatment. This activity is blocked by pretreatment of the cells with the MEK inhibitors PD98059 and UO126. The mechanism by which ERKs-1 and -2 are activated by estrogen appears to be independent of c-Src since the effects of estrogen on PRL gene expression are not affected by herbimycin A or PP1 administration. c-Raf-1 may be involved in the effects of E2 because estrogen causes the rapid and transient tyrosine phosphorylation of c-Raf-1. The ER antagonist ICI 182,780 blocks both ERK-1 and ERK-2 activation in addition to PRL protein and mRNA, implying a central role for the classical ER in the activation of the MAPK pathway resulting in PRL gene expression.     

Kinase Suppressor of Ras Forms a Multiprotein Signaling Complex and Modulates MEK Localization

Genetic screens for modifiers of activated Ras phenotypes have identified a novel protein, kinase suppressor of Ras (KSR), which shares significant sequence homology with Raf family protein kinases. Studies using Drosophila melanogaster and Caenorhabditis elegans predict that KSR positively regulates Ras signaling; however, the function of mammalian KSR is not well understood. We show here that two predicted kinase-dead mutants of KSR retain the ability to complement ksr-1 loss-of-function alleles in C. elegans, suggesting that KSR may have physiological, kinase-independent functions. Furthermore, we observe that murine KSR forms a multimolecular signaling complex in human embryonic kidney 293T cells composed of HSP90, HSP70, HSP68, p50(CDC37), MEK1, MEK2, 14-3-3, and several other, unidentified proteins. Treatment of cells with geldanamycin, an inhibitor of HSP90, decreases the half-life of KSR, suggesting that HSPs may serve to stabilize KSR. Both nematode and mammalian KSRs are capable of binding to MEKs, and three-point mutants of KSR, corresponding to C. elegans loss-of-function alleles, are specifically compromised in MEK binding. KSR did not alter MEK activity or activation. However, KSR-MEK binding shifts the apparent molecular mass of MEK from 44 to >700 kDa, and this results in the appearance of MEK in membrane-associated fractions. Together, these results suggest that KSR may act as a scaffolding protein for the Ras-mitogen-activated protein kinase pathway.     

CK2 Is a component of the KSR1 scaffold complex that contributes to Raf kinase activation

Kinase Suppressor of Ras (KSR) is a molecular scaffold that interacts with the core kinase components of the ERK cascade, Raf, MEK, and ERK and provides spatial and temporal regulation of Ras-dependent ERK cascade signaling. In this report, we identify the heterotetrameric protein kinase, casein kinase 2 (CK2), as a new KSR1-binding partner. Moreover, we find that the KSR1/CK2 interaction is required for KSR1 to maximally facilitate ERK cascade signaling and contributes to the regulation of Raf kinase activity. Binding of the CK2 holoenzyme is constitutive and requires the basic surface region of the KSR1 atypical C1 domain. Loss of CK2 binding does not alter the membrane translocation of KSR1 or its interaction with ERK cascade components; however, disruption of the KSR1/CK2 interaction or inhibition of CK2 activity significantly reduces the growth-factor-induced phosphorylation of C-Raf and B-Raf on the activating serine site in the negative-charge regulatory region (N-region). This decrease in Raf N-region phosphorylation further correlates with impaired Raf, MEK, and ERK activation. These findings identify CK2 as a novel component of the KSR1 scaffolding complex that facilitates ERK cascade signaling by functioning as a Raf family N-Region kinase.