Phosphorylation-regulated nucleocytoplasmic trafficking of internalized fibroblast growth factor-1

Fibroblast growth factor-1 (FGF-1), which stimulates cell growth, differentiation, and migration, is capable of crossing cellular membranes to reach the cytosol and the nucleus in cells containing specific FGF receptors. The cell entry process can be monitored by phosphorylation of the translocated FGF-1. We present evidence that phosphorylation of FGF-1 occurs in the nucleus by protein kinase C (PKC)delta. The phosphorylated FGF-1 is subsequently exported to the cytosol. A mutant growth factor where serine at the phosphorylation site is exchanged with glutamic acid, to mimic phosphorylated FGF-1, is constitutively transported to the cytosol, whereas a mutant containing alanine at this site remains in the nucleus. The export can be blocked by leptomycin B, indicating active and receptor-mediated nuclear export of FGF-1. Thapsigargin, but not leptomycin B, prevents the appearance of active PKCdelta in the nucleus, and FGF-1 is in this case phosphorylated in the cytosol. Leptomycin B increases the amount of phosphorylated FGF-1 in the cells by preventing dephosphorylation of the growth factor, which seems to occur more rapidly in the cytoplasm than in the nucleus. The nucleocytoplasmic trafficking of the phosphorylated growth factor is likely to play a role in the activity of internalized FGF-1.     

Exogenously added fibroblast growth factor 2 (FGF-2) to NIH3T3 cells interacts with nuclear ribosomal S6 kinase 2 (RSK2) in a cell cycle-dependent manner

Fibroblast growth factor 2 (FGF-2) has been detected in the nuclei of many tissues and cell lines. Here we demonstrate that FGF-2 added exogenously to NIH3T3 cells enters the nucleus and interacts with the nuclear active 90-kDa ribosomal S6 kinase 2 (RSK2) in a cell cycle-dependent manner. By using purified proteins, FGF-2 is shown to directly interact through two separate domains with two RSK2 domains on both sides of the hydrophobic motif, namely the NH2-terminal kinase domain (residues 360-381) by amino acid Ser-117 and the COOH-terminal kinase domain (residues 388-400) by amino acids Leu-127 and Lys-128. Moreover, this interaction leads to maintenance of the sustained activation of RSK2 in G1 phase of the cell cycle. FGF-2 mutants (FGF-2 S117A, FGF-2 L127A, and FGF-2 K128A) that fail to interact in vitro with RSK2 fail to maintain a sustained RSK2 activity in vivo.     

The cyclin-dependent kinase inhibitor p27Kip1 is stabilized in G(0) by Mirk/dyrk1B kinase

Elevated levels of the cyclin-dependent kinase (CDK) inhibitor p27 block the cell in G(0)/G(1) until mitogenic signals activate G(1) cyclins and initiate proliferation. Post-translational regulation of p27 by different phosphorylation events is critical in allowing cells to proceed through the cell cycle. We now demonstrate that the arginine-directed kinase, Mirk/dyrk1B, is maximally active in G(0) in NIH3T3 cells, when it stabilizes p27 by phosphorylating it at Ser-10. The phospho-mimetic mutant p27-S10D was more stable, and the non-phosphorylatable mutant p27-S10A was less stable than wild-type when expressed in G(0)-arrested cells. Following phosphorylation by Mirk, p27 remains a functional CDK inhibitor, capable of binding to CDK2. Mirk did not induce the translocation of p27 from the nucleus in G(0), but instead co-localized with nuclear p27. Depletion of Mirk by RNA interference decreased the phosphorylation of p27 at Ser-10 and the stability of endogenous p27. RNA(i) to Mirk increased cell entry from G(0) into G(1) as shown by increased expression of proliferating cell nuclear antigen and decreased expression of p27. These data suggest a model in which Mirk increases the amount of nuclear p27 by stabilizing it during G(0) when Mirk is most abundant. Mitogen stimulation then causes cells to enter G(1), reduces Mirk levels (Deng, X., Ewton, D., Pawlikowski, B., Maimone, M., and Friedman, E. (2003) J. Biol. Chem. 278, 41347-41354), and initiates the translocation of p27 to the cytoplasm. In addition, depletion of Mirk by RNA(i) in postmitotic C2C12 myoblasts decreased protein but not mRNA levels of p27, suggesting that stabilization of p27 by Mirk also occurs during differentiation.     

Structure-function studies of FGF-1: Dissociation and partial reconstitution of certain of its biological activities

We reported previously that the mitogenic activities of FGF-1 (acidic FGF) could be dissociated from its receptor-binding activities by site-directed mutagenesis of lysine 132 to a glutamic acid. Although the mutant FGF-1 protein binds to the high-affinity tyrosinekinase receptors, stimulates tyrosine-kinase activity, and promotes expression of immediate-early genes, it is not mitogenic for a variety of tested cell lines. Interestingly, the mutant FGF-1 is capable of other functions associated with the wild-type protein such as promotion of mesoderm formation in Xenopus animal caps. The mutant exhibits a reduced apparent affinity for heparin-Sepharose compared to the wild-type protein. The relationship between the reduced heparin affinity and lack of mitogenic activity of this mutant is not clear. Recent data indicates the relationship is not as simple as reduced stability of the protein. When NIH 3T3 cells are transfected with expression vectors encoding either wild-type or mutant FGF-1, a transformed phenotype can be seen in cells overexpressing the wild-type FGF-1, whereas cells overexpressing mutant FGF-1 appear normal. Analysis of lysates of these cells indicates that a tyrosine-kinase cascade, distinct from that associated with the high-affinity cell surface receptors, has been activated in the wild-type transfected cells but not in the mutant transfected cells. Although both transfected cell lines contain FGF-1 cell surface receptors as judged by crosslinking studies, the wild-type transfects are refractory to exogenous FGF-1, whereas the mutant transfectants respond normally. Together these results support an intracellular role of wild-type FGF-1 in mediating certain of its functions. In addition, they demonstrate that certain functions of the growth factor can be dissociated at the structural level. Additional mutagenesis studies have resulted in the identification of mutants with heparin-binding or mitogenic deficiencies that do not correlate as well as those of the 132 mutant. It appears that the inactivity of the lysine 132 mutant is related, in part, to cysteine 131. © 1994 Wiley-Liss, Inc.     

Live-cell fluorescence imaging reveals the dynamics of protein kinase CK2 individual subunits

Protein kinase CK2 is a multifunctional enzyme which has long been described as a stable heterotetrameric complex resulting from the association of two catalytic (alpha or alpha') and two regulatory (beta) subunits. To track the spatiotemporal dynamics of CK2 in living cells, we fused its catalytic alpha and regulatory beta subunits with green fluorescent protein (GFP). Both CK2 subunits contain nuclear localization domains that target them independently to the nucleus. Imaging of stable cell lines expressing low levels of GFP-CK2alpha or GFP-CK2beta revealed the existence of CK2 subunit subpopulations exhibiting differential dynamics. Once in the nucleus, they diffuse randomly at different rates. Unlike CK2beta, CK2alpha can shuttle, showing the dynamic nature of the nucleocytoplasmic trafficking of the kinase. When microinjected in the cytoplasm, the isolated CK2 subunits are rapidly translocated into the nucleus, whereas the holoenzyme complex remains in this cell compartment, suggesting an intramolecular masking of the nuclear localization sequences that suppresses nuclear accumulation. However, binding of FGF-2 to the holoenzyme triggers its nuclear translocation. Since the substrate specificity of CK2alpha is dramatically changed by its association with CK2beta, the control of the nucleocytoplasmic distribution of each subunit may represent a unique potential regulatory mechanism for CK2 activity.     

Ligand dependent and independent internalization and nuclear translocation of fibroblast growth factor (FGF) receptor 1

Basic fibroblast growth factor (FGF-2) is one of the prototype members of a rapidly expanding family of polypeptides. FGF-2 acts on cells via a dual-receptor system consisting of high-affinity tyrosine kinase receptors (FGFR) and low-affinity receptors comprised of heparan sulfate proteoglycans. Following ligand binding and subsequent internalization, both FGF-2 and FGFR1 are translocated to the nucleus where they have activities distinct from those expressed at the cell surface. Despite the growing number of growth factors and receptors shown to translocate to the nucleus, little is known about the mechanisms of internalization and translocation and how these processes are regulated. In the studies reported in this paper, we examined the roles of clathrin-dependent and -independent endocytosis in the uptake of FGFR1 and one of its ligands, FGF-2. While the uptake of FGF-2 occurred at least partly by a caveolar-dependent mechanism, that of FGFR1 was independent of both caveolae and coated pits. Surprisingly, neither the uptake of FGF-2 nor FGFR1 required the activity of the receptor tyrosine kinase. In addition, we identified a cell cycle-dependent pathway of FGFR1 nuclear translocation that appears to be independent of ligand binding.     

Structural requirements of FGF-1 for receptor binding and translocation into cells

FGF-1 binds to and activates specific transmembrane receptors (FGFRs) and is subsequently internalized and translocated to the interior of the cell. To elucidate the role of the receptor in the translocation process, we studied the effects of the elimination of distinct sites of the ligand-receptor interaction. On the basis of the structure of the FGF-1-FGFR1 complex, we substituted four key amino acid residues of FGF-1 from the FGF-receptor binding site with alanines, constructing four point mutants and one double mutant. We determined by in vivo assays in NIH 3T3 cells the ability of the mutants to bind to specific FGF receptors, to stimulate DNA synthesis, and to activate downstream signaling pathways. We found that correct binding to the receptor is necessary for optimal stimulation of DNA synthesis. All four single mutants became phosphorylated to different extents, indicating that they were translocated to the cytosol/nucleus with varying efficiency. This indicates that despite a low affinity for FGFR, translocation to the cytosol/nucleus can still occur. However, simultaneous substitution in two of the positions led to a total loss of biological activity of the growth factor and prevented its internalization, implying that there is only one strongly receptor-dependent, productive way of translocating FGF-1. We also found that the process of translocation did not correlate with the thermal stability of the protein. Additionally, we observed a clear negative correlation between the stability of the FGF-1 mutants and the efficiency of their phosphorylation, which strongly suggests that protein kinases prefer the unfolded state of the protein substrate.     

Two nuclear localization signals required for transport from the cytosol to the nucleus of externally added FGF-1 translocated into cells

Externally added FGF-1 is transported into the nucleus of cells. It was earlier shown that FGF-1 contains an N-terminal nuclear localization signal (NLS) implicated in the stimulation of DNA synthesis. We here provide evidence that FGF-1 contains a second putative NLS (NLS2), which is located near the C-terminus. It is a bipartite NLS consisting of two clusters of lysines separated by a spacer of 10 amino acids. A fusion protein of GFP and the bipartite NLS was more efficiently transported into the nucleus than GFP alone, indicating that it can act as an NLS in the living cell. FGF-1 mutated in the N-terminal NLS (NLS1) or in the first cluster of the bipartite NLS2 bound to heparin and FGF receptors and activated downstream signaling similarly to the wild-type growth factor. Mutations in the second cluster of NLS2 resulted in impaired interaction with heparin and reduced stability. When radiolabeled FGF-1 with mutated NLS1 or the first lysine cluster of NLS2 was added to NIH/3T3 cells, it was translocated into the cytosol, but not transported efficiently to the nucleus. Phosphorylation of FGF-1 occurs normally in the nucleus, and while wild-type FGF-1 was phosphorylated after addition to cells, the NLS mutants were not. It therefore appears that both NLS1 and NLS2 are important for efficient transport of FGF-1 to the nucleus. Stimulation of DNA synthesis by FGF-1 with mutations in both NLSs was reduced considerably indicating that efficient transport to the nucleus may be involved in the stimulation of DNA synthesis.     

Analysis of endogenous and exogenous nuclear translocation of fibroblast growth factor-1 in NIH 3T3 cells.

Nuclear localization of fibroblast growth factors (FGF) have been reported by many laboratories. We demonstrate here that FGF-1, the precursor for acidic FGF contains a putative nuclear translocation sequence (NTS) NYKKPKL, which is able to direct the expression of the bacterial beta galactosidase (beta gal) gene to the nucleus of transfected NIH 3T3 cells. However, this NTS is unable to target either FGF-1 itself or a FGF-1-beta gal fusion protein into the nucleus, suggesting that FGF-1 may contain an additional sequence which prevents endogenously expressed FGF-1 from being translocated into the nucleus. Indeed, when FGF-1 was fused to the NTS derived from the yeast histone 2B gene, the chimeric construct also failed to be transported into the nucleus either by itself or as a beta gal fusion protein. Interestingly, when 125I-FGF-1 was used to stimulate quiescent NIH 3T3 cells, a significant amount of internalized 125I-FGF-1 (approximately 10%) was found within the nucleus and the nuclear localization of FGF-1 through the exogenous pathway could be significantly reduced by suramin, an inhibitor of the interaction of FGF-1 with its receptor. These data suggest that while FGF-1 contains a NTS, nuclear translocation requires an exogenous and not an endogenous pathway.     

Nuclear accumulation of fibroblast growth factor receptors is regulated by multiple signals in adrenal medullary cells.

In an effort to determine the localization of fibroblast growth factor (FGF) receptors (FGFR) that could mediate the intracellular action of FGF-2, we discovered the presence of high-affinity. FGF-2 binding sites in the nuclei of bovine adrenal medullary cells (BAMC). Western blot analysis demonstrated the presence of 103-, 118-, and 145-kDa forms of FGFR1 in nuclei isolated from BAMC. 125I-FGF-2 cross-linking to nuclear extracts followed by FGFR1 immunoprecipitation showed that FGFR1 can account for the nuclear FGF-2 binding sites. Nuclear FGFR1 has kinase activity and undergoes autophosphorylation. Immunocytochemistry with the use of confocal and electron microscopes demonstrated the presence of FGFR1 within the nuclear interior. Nuclear subfractionation followed by Western blot or immunoelectron microscopic analysis showed that the nuclear FGFR1 is contained in the nuclear matrix and the nucleoplasm. Agents that induce translocation of endogenous FGF-2 to the nucleus (forskolin, carbachol, or angiotensin II) increased the intranuclear accumulation of FGFR1. This accumulation was accompanied by an overall increase in FGF-2-inducible tyrosine kinase activity. Our findings suggest a novel mode for growth factor action whereby growth factor receptors translocate to the nucleus in parallel with their ligand and act as direct mediators of nuclear responses to cell stimulation.     

Mirk/Dyrk1B mediates survival during the differentiation of C2C12 myoblasts

The kinase Mirk/dyrk1B is essential for the differentiation of C2C12 myoblasts. Mirk reinforces the G0/G1 arrest state in which differentiation occurs by directly phosphorylating and stabilizing p27(Kip1) and destabilizing cyclin D1. We now demonstrate that Mirk is anti-apoptotic in myoblasts. Knockdown of endogenous Mirk by RNA interference activated caspase 3 and decreased myoblast survival by 75%, whereas transient overexpression of Mirk increased cell survival. Mirk exerts its anti-apoptotic effects during muscle differentiation at least in part through effects on the cell cycle inhibitor and pro-survival molecule p21(Cip1). Overexpression and RNA interference experiments demonstrated that Mirk phosphorylates p21 within its nuclear localization domain at Ser-153 causing a portion of the typically nuclear p21 to localize in the cytoplasm. Phosphomimetic GFP-p21-S153D was pancellular in both cycling C2C12 myoblasts and NIH3T3 cells. Endogenous Mirk in myotubes and overexpressed Mirk in NIH3T3 cells were able to cause the pancellular localization of wild-type GFP-p21 but not the nonphosphorylatable mutant GFP-p21-S153A. Translocation to the cytoplasm enables p21 to block apoptosis through inhibitory interaction with pro-apoptotic molecules. Phosphomimetic p21-S153D was more effective than wild-type p21 in blocking the activation of caspase 3. Transient expression of p21-S153D also increased myoblast viability in colony forming assays, whereas the p21-S153A mutant had no effect. This Mirk-dependent change in p21 intracellular localization is a natural part of myoblast differentiation. Endogenous p21 localized exclusively to the nuclei of proliferating myoblasts but was also found in the cytoplasm of post-mitotic multinucleated myotubes and adult human skeletal myofibers.     

Cell cycle dependent regulation of protein kinase CK2 signaling to the nuclear matrix

Protein kinase CK2 is a ubiquitous protein serine/threonine kinase that is involved in cell growth and proliferation as well as suppression of apoptosis. Several studies have suggested that the kinase plays a role in cell cycle progression; however, changes in enzyme activity during phases of cell cycle have not been detected. Nuclear matrix is a key locus for CK2 signaling in the nucleus. We therefore examined CK2 signaling to the nuclear matrix in distinct phases of cell cycle by employing synchronized ALVA-41 prostate cancer cells. Removal of serum from the culture medium resulted in G0/G1 arrest, and a reduction in the nuclear matrix-associated CK2 activity which was rapidly reversed on addition of serum. Arresting the cells in G(0)/G(1) phase with hydroxyurea and subsequent release to S phase by serum gave similar results. Cells arrested in the G(2)/M phase by treatment with nocodazole demonstrated an extensive reduction in the nuclear matrix-associated CK2 which was reversed rapidly on addition of serum. Changes in the immunoreactive CK2 protein were concordant with the activity data reflecting a dynamic trafficking of the kinase in distinct phases of cell cycle. Under the same conditions, CK2 activity in total cellular lysate remained essentially unaltered. These results provide the first direct evidence of discrete modulations of CK2 in the nuclear matrix during the cell cycle progression. Inducible overexpression of CK2 in CHO cells yielded only a modest increase in CK2 activity even though a significant increase in expression was apparent at the level of CK2 alpha-specific message. Stably transfected ALVA-41 cells, however, did not show a significant change in CK2 levels despite increased expression at the message level. Not surprisingly, both types of the stably transfected cells failed to show any alteration in cell cycle progression. Distribution of the CK2 activity in the cytosolic versus nuclear matrix fractions in normal cells appears to be different from that in the cancer cells such that the ratio of nuclear matrix to cytosolic activity is much higher in the latter. Considering that nuclear matrix is central to several nuclear functions, this pattern of intracellular distribution of CK2 may have implications for its role in the oncogenic process. Published 2003 Wiley-Liss, Inc.     

Integrative nuclear FGFR1 signaling (INFS) pathway mediates activation of the tyrosine hydroxylase gene by angiotensin II,depolarization and protein kinase C

The integrative nuclear FGFR1 signaling (INFS) pathway functions in association with cellular growth, differentiation, and regulation of gene expression, and is activated by diverse extracellular signals. Here we show that stimulation of angiotensin II (AII) receptors, depolarization, or activation protein kinase C (PKC) or adenylate cyclase all lead to nuclear accumulation of fibroblast growth factor 2 (FGF-2) and FGFR1, association of FGFR1 with splicing factor-rich domains, and activation of the tyrosine hydroxylase (TH) gene promoter in bovine adrenal medullary cells (BAMC). The up-regulation of endogenous TH protein or a transfected TH promoter-luciferase construct by AII, veratridine, or PMA (but not by forskolin) is abolished by transfection with a dominant negative FGFR1TK-mutant which localizes to the nucleus and plasma membrane, but not by extracellularly acting FGFR1 antagonists suramin and inositolhexakisphosphate (IP6). Mechanism of TH gene activation by FGF-2 and FGFR1 was further investigated in BAMC and human TE671 cultures. TH promoter was activated by co-transfected HMW FGF-2 (which is exclusively nuclear) but not by cytoplasmic FGF-1 or extracellular FGFs. Promoter transactivation by HMWFGF-2 was accompanied by an up-regulation of FGFR1 specifically in the cell nucleus and was prevented FGFR1(TK-) but not by IP6 or suramin. The TH promoter was also transactivated by co-transfected wild-type FGFR1, which localizes to both to the nucleus and the plasma membrane, and by an exclusively nuclear, soluble FGFR1(SP-/NLS) mutant with an inserted nuclear localization signal. Activation of the TH promoter by nuclear FGFR1 and FGF-2 was mediated through the cAMP-responsive element (CRE) and was associated with induction of CREB- and CBP/P-300-containing CRE complexes. We propose a new model for gene regulation in which nuclear FGFR1 acts as a mediator of CRE transactivation by AII, cell depolarization, and PKC.     

A distinct basic fibroblast growth factor (FGF-2)/FGF receptor interaction distinguishes urokinase-type plasminogen activator induction from mitogenicity in endothelial cells.

Basic fibroblast growth factor (FGF-2) induces cell proliferation and urokinase-type plasminogen activator (uPA) production in fetal bovine aortic endothelial GM 7373 cells. In the present paper we investigated the role of the interaction of FGF-2 with tyrosine-kinase (TK) FGF receptors (FGFRs) in mediating uPA up-regulation in these cells. The results show that FGF-2 antagonists suramin, protamine, heparin, the synthetic peptide FGF-2(112-155), and a soluble form of FGFR-1 do not inhibit FGF-2-mediated uPA up-regulation at concentrations that affect growth factor binding to cell surface receptors and mitogenic activity. In contrast, tyrosine phosphorylation inhibitors and overexpression of a dominant negative TK- mutant of FGFR-1 abolish the uPA-inducing activity of FGF-2, indicating that FGFR and its TK activity are essential in mediating uPA induction. Accordingly, FGF-2 induces uPA up-regulation in Chinese hamster ovary cells transfected with wild-type FGFR-1, -2, -3, or -4 but not with TK- FGFR-1 mutant. Small unilamellar phosphatidyl choline:cholesterol vesicles loaded with FGF-2 increased uPA production in GM 7373 cells in the absence of a mitogenic response. Liposome-encapsulated FGF-2 showed a limited but significant capacity, relative to free FGF-2, to interact with FGFR both at 4 degrees C and 37 degrees C and to be internalized within the cell. uPA up-regulation by liposome-encapsulated FGF-2 was quenched by neutralizing anti-FGF-2 antibodies, indicating that the activity of liposome-delivered FGF-2 is mediated by an extracellular action of the growth factor. Taken together, the data indicate that a distinct interaction of FGF-2 with FGFR, quantitatively and/or qualitatively different from the one that leads to mitogenicity, is responsible for the uPA-inducing activity of the growth factor.     

Fibroblast growth factor-2

Fibroblast growth factor-2 (FGF-2) is a member of a large family of proteins that bind heparin and heparan sulfate and modulate the function of a wide range of cell types. FGF-2 stimulates the growth and development of new blood vessels (angiogenesis) that contribute to the pathogenesis of several diseases (i.e. cancer, atherosclerosis), normal wound healing and tissue development. FGF-2 contains a number of basic residues (pI 9.6) and consists of 12 anti-parallel beta-sheets organized into a trigonal pyrimidal structure. FGF-2 binds to four cell surface receptors expressed as a number of splice variants. Many of the biological activities of FGF-2 have been found to depend on its receptor's intrinsic tyrosine kinase activity and second messengers such as the mitogen activated protein kinases. However, considerable evidence suggest that intracellular FGF-2 might have a direct biological role particularly within the nucleus. In addition, heparan sulfate proteoglycans have been demonstrated to enhance and inhibit FGF-2 activity. The possibility that FGF-2 activity can be manipulated through alterations in heparan sulfate-binding is currently being exploited in the development of clinical applications aimed at modulating either endogenous or administered FGF-2 activity.     

碱性成纤维细胞生长因子研究进展

碱性成纤维细胞生长因子是细胞生长和分化的重要调节因子,具有促血管生成、细胞增殖、细胞趋化、细胞迁移等活性,在细胞分化和机体发育过程中发挥重要作用。碱性成纤维细胞生长因子通过与细胞膜表面的特异性配体结合,进而引发细胞内的一系列级联反应,从而产生各种生物学效应。本文对碱性成纤维细胞生长因子的生物学基础、信号转导、生物学功能以及临床应用研究进展作一综述。     

Activation and targeting of mitogen-activated protein kinases by G-protein-coupled receptors

Over the past decade, it has become apparent that many G-protein-coupled receptors (GPCRs) generate signals that control cellular differentiation and growth, including stimulation of Ras family GTPases and activation of mitogen-activated protein (MAP) kinase pathways. The mechanisms that GPCRs use to control the activity of MAP kinases vary between receptor and cell type but fall broadly into one of three categories: signals initiated by classical G protein effectors, e.g., protein kinase (PK)A and PKC, signals initiated by cross-talk between GPCRs and classical receptor tyrosine kinases, e.g., "transactivation" of epidermal growth factor (EGF) receptors, and signals initiated by direct interaction between beta-arrestins and components of the MAP kinase cascade, e.g., beta-arrestin "scaffolds". While each of these pathways results in increased cellular MAP kinase activity, emerging data suggest that they are not functionally redundant. MAP kinase activation occurring via PKC-dependent pathways and EGF receptor transactivation leads to nuclear translocation of the kinase and stimulates cell proliferation, while MAP kinase activation via beta-arrestin scaffolds primarily increases cytosolic kinase activity. By controlling the spatial and temporal distribution of MAP kinase activity within the cell, the consequences of GPCR-stimulated MAP kinase activation may be determined by the mechanism by which they are activated.     

Regulation of c-myc expression by PDGF through Rho GTPases

Src family protein-tyrosine kinases have a central role in several biological functions, including cell adhesion and spreading, chemotaxis, cell cycle progression, differentiation and apoptosis. Surprisingly, these kinases also participate in mitogenic signalling by receptors that themselves exhibit an intrinsic protein-tyrosine kinase activity, including those for platelet-derived growth factor (PDGF), epidermal growth factor and colony-stimulating factor-1. Indeed, Src kinases are strictly required for the nuclear expression of the c-myc proto-oncogene and thus for DNA synthesis in response to PDGF. However, the nature of the signalling pathways by which Src kinases participate in the induction of c-myc expression by tyrosine kinase receptors is still unknown. Here we show that PDGF enhances c-myc expression and stimulates the c-myc promoter in a Src-dependent manner, and that neither Ras nor the mitogen-activated protein kinase pathway mediate these effects. In contrast, we present evidence that PDGF stimulates Vav2 through Src, thereby initiating the activation of a Rac-dependent pathway that controls the expression of the c-myc proto-oncogene.     

Nuclear export of S6K1 II is regulated by protein kinase CK2 phosphorylation at Ser-17

Ribosomal S6 kinases (S6Ks) are principal players in the regulation of cell growth and energy metabolism. Signaling via phosphatidylinositol 3-kinase and mammalian target of rapamycin pathways mediates the activation of S6K in response to various mitogenic stimuli. The family of S6Ks consists of two forms, S6K1 and -2, that have cytoplasmic and nuclear splicing variants, S6K1 II and S6K1 I, respectively. Nuclear-cytoplasmic shuttling of both isoforms induced by mitogenic stimuli has been reported recently. Here we present the identification of protein kinase CK2 (CK2) as a novel binding and regulatory partner for S6K1 II. The interaction between S6K1 II and CK2beta regulatory subunit was initially identified in a yeast two-hybrid screen and further confirmed by co-immunoprecipitation of transiently expressed and endogenous proteins. The interaction between S6K1 II and CK2 was found to occur in serum-starved and serum-stimulated cells. In addition, we found that S6K1 II is a substrate for CK2. The localization of the CK2 phosphorylation site was narrowed down to Ser-17 in S6K1 II. Mutational analysis and the use of phosphospecific antibody indicate that Ser-17 is a major in vitro and in vivo phosphorylation site for CK2. Functional studies reveal that, in contrast to the wild type kinase, the phosphorylation-mimicking mutant of S6K1 II (S17E) retains its cytoplasmic localization in serum-stimulated cells. Treatment of cells with the nuclear export inhibitor leptomycin B revealed that the S17E mutant accumulates in the nucleus to the same extent as S6K1 II wild type. These results indicate that nuclear import of the S17E mutant is not affected, although the export is significantly enhanced. We also provide evidence that nuclear export of S6K1 is mediated by a CRM1-dependent mechanism. Taken together, this study establishes a functional link between S6K1 II and CK2 signaling, which involves the regulation of S6K1 II nuclear export by CK2-mediated phosphorylation of Ser-17.