RhoG GTPase Controls a Pathway That Independently Activates Rac1 and Cdc42Hs

RhoG is a member of the Rho family of GTPases that shares 72% and 62% sequence identity with Rac1 and Cdc42Hs, respectively. We have expressed mutant RhoG proteins fused to the green fluorescent protein and analyzed subsequent changes in cell surface morphology and modifications of cytoskeletal structures. In rat and mouse fibroblasts, green fluorescent protein chimera and endogenous RhoG proteins colocalize according to a tubular cytoplasmic pattern, with perinuclear accumulation and local concentration at the plasma membrane. Constitutively active RhoG proteins produce morphological and cytoskeletal changes similar to those elicited by a simultaneous activation of Rac1 and Cdc42Hs, i.e., the formation of ruffles, lamellipodia, filopodia, and partial loss of stress fibers. In addition, RhoG and Cdc42Hs promote the formation of microvilli at the cell apical membrane. RhoG-dependent events are not mediated through a direct interaction with Rac1 and Cdc42Hs targets such as PAK-1, POR1, or WASP proteins but require endogenous Rac1 and Cdc42Hs activities: coexpression of a dominant negative Rac1 impairs membrane ruffling and lamellipodia but not filopodia or microvilli formation. Conversely, coexpression of a dominant negative Cdc42Hs only blocks microvilli and filopodia, but not membrane ruffling and lamellipodia. Microtubule depolymerization upon nocodazole treatment leads to a loss of RhoG protein from the cell periphery associated with a reversal of the RhoG phenotype, whereas PDGF or bradykinin stimulation of nocodazole-treated cells could still promote Rac1- and Cdc42Hs-dependent cytoskeletal reorganization. Therefore, our data demonstrate that RhoG controls a pathway that requires the microtubule network and activates Rac1 and Cdc42Hs independently of their growth factor signaling pathways.

Integrin-mediated Signaling Events in Human Endothelial Cells

Vascular endothelial cells are important in a variety of physiological and pathophysiological processes. The growth and functions of vascular endothelial cells are regulated both by soluble mitogenic and differentiation factors and by interactions with the extracellular matrix; however, relatively little is known about the role of the matrix. In the present study, we investigate whether integrin-mediated anchorage to a substratum coated with the extracellular matrix protein fibronectin regulates growth factor signaling events in human endothelial cells. We show that cell adhesion to fibronectin and growth factor stimulation trigger distinct initial tyrosine phosphorylation events in endothelial cells. Thus, integrin-dependent adhesion of endothelial cells leads to tyrosine phosphorylation of both focal adhesion kinase and paxillin, but not of several growth factor receptors. Conversely, EGF stimulation causes receptor autophosphorylation, with no effect on focal adhesion kinase or paxillin tyrosine phosphorylation. Adhesion to fibronectin, in the absence of growth factors, leads to activation of MAPK. In addition, adhesion to fibronectin also potentiates growth factor signaling to MAPK. Thus, polypeptide growth factor activation of MAPK in anchored cells is far more effective than in cells maintained in suspension. Other agonists known to activate MAPK were also examined for their ability to activate MAPK in an anchorage-dependent manner. The neuropeptide bombesin, the bioactive lipid lysophosphatidic acid (LPA), and the cytokine tumor necrosis factor α, which signal through diverse mechanisms, were all able to activate MAPK to a much greater degree in fibronectin-adherent cells than in suspended cells. In addition, tumor necrosis factor α activation of c-Jun kinase (JNK) was also much more robust in anchored cells. Together, these data suggest a cooperation between integrins and soluble mitogens in efficient propagation of signals to downstream kinases. This cooperation may contribute to anchorage dependence of mitogenic cell cycle progression.

Functional Characterization of the Interaction of Ste50p with Ste11p MAPKKK in Saccharomyces cerevisiae

The Saccharomyces cerevisiae Ste11p protein kinase is a homologue of mammalian MAPK/extracellular signal-regulated protein kinase kinase kinases (MAPKKKs or MEKKs) as well as the Schizosaccharomyces pombe Byr2p kinase. Ste11p functions in several signaling pathways, including those for mating pheromone response and osmotic stress response. The Ste11p kinase has an N-terminal domain that interacts with other signaling molecules to regulate Ste11p function and direct its activity in these pathways. One of the Ste11p regulators is Ste50p, and Ste11p and Ste50p associate through their respective N-terminal domains. This interaction relieves a negative activity of the Ste11p N terminus, and removal of this negative function is required for Ste11p function in the high-osmolarity glycerol (HOG) pathway. The Ste50p/Ste11p interaction is also important (but not essential) for Ste11p function in the mating pathway; in this pathway binding of the Ste11p N terminus with both Ste50p and Ste5p is required, with the Ste5p association playing the major role in Ste11p function. In vitro, Ste50p disrupts an association between the catalytic C terminus and the regulatory N terminus of Ste11p. In addition, Ste50p appears to modulate Ste11p autophosphorylation and is itself a substrate of the Ste11p kinase. Therefore, both in vivo and in vitro data support a role for Ste50p in the regulation of Ste11p activity.

Radiation-induced Release of Transforming Growth Factor α Activates the Epidermal Growth Factor Receptor and Mitogen-activated Protein Kinase Pathway in Carcinoma Cells, Leading to Increased Proliferation and Protection from Radiation-induced Cell Death

Exposure of A431 squamous and MDA-MB-231 mammary carcinoma cells to ionizing radiation has been associated with short transient increases in epidermal growth factor receptor (EGFR) tyrosine phosphorylation and activation of the mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal kinase (JNK) pathways. Irradiation (2 Gy) of A431 and MDA-MB-231 cells caused immediate primary activations (0–10 min) of the EGFR and the MAPK and JNK pathways, which were surprisingly followed by later prolonged secondary activations (90–240 min). Primary and secondary activation of the EGFR was abolished by molecular inhibition of EGFR function. The primary and secondary activation of the MAPK pathway was abolished by molecular inhibition of either EGFR or Ras function. In contrast, molecular inhibition of EGFR function abolished the secondary but not the primary activation of the JNK pathway. Inhibition of tumor necrosis factor α receptor function by use of neutralizing monoclonal antibodies blunted primary activation of the JNK pathway. Addition of a neutralizing monoclonal antibody versus transforming growth factor α (TGFα) had no effect on the primary activation of either the EGFR or the MAPK and JNK pathways after irradiation but abolished the secondary activation of EGFR, MAPK, and JNK. Irradiation of cells increased pro-TGFα cleavage 120–180 min after exposure. In agreement with radiation-induced release of a soluble factor, activation of the EGFR and the MAPK and JNK pathways could be induced in nonirradiated cells by the transfer of media from irradiated cells 120 min after irradiation. The ability of the transferred media to cause MAPK and JNK activation was blocked when media were incubated with a neutralizing antibody to TGFα. Thus radiation causes primary and secondary activation of the EGFR and the MAPK and JNK pathways in autocrine-regulated carcinoma cells. Secondary activation of the EGFR and the MAPK and JNK pathways is dependent on radiation-induced cleavage and autocrine action of TGFα. Neutralization of TGFα function by an anti-TGFα antibody or inhibition of MAPK function by MEK1/2 inhibitors (PD98059 and U0126) radiosensitized A431 and MDA-MB-231 cells after irradiation in apoptosis, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), and clonogenic assays. These data demonstrate that disruption of the TGFα–EGFR–MAPK signaling module represents a strategy to decrease carcinoma cell growth and survival after irradiation.

Two Distinct Mechanisms Control the Accumulation of Cyclin B1 and Mos in Xenopus Oocytes in Response to Progesterone

Progesterone-induced meiotic maturation of Xenopus oocytes requires the synthesis of new proteins, such as Mos and cyclin B. Synthesis of Mos is thought to be necessary and sufficient for meiotic maturation; however, it has recently been proposed that newly synthesized proteins binding to p34cdc2 could be involved in a signaling pathway that triggers the activation of maturation-promoting factor. We focused our attention on cyclin B proteins because they are synthesized in response to progesterone, they bind to p34cdc2, and their microinjection into resting oocytes induces meiotic maturation. We investigated cyclin B accumulation in response to progesterone in the absence of maturation-promoting factor–induced feedback. We report here that the cdk inhibitor p21cip1, when microinjected into immature Xenopus oocytes, blocks germinal vesicle breakdown induced by progesterone, by maturation-promoting factor transfer, or by injection of okadaic acid. After microinjection of p21cip1, progesterone fails to induce the activation of MAPK or p34cdc2, and Mos does not accumulate. In contrast, the level of cyclin B1 increases normally in a manner dependent on down-regulation of cAMP-dependent protein kinase but independent of cap-ribose methylation of mRNA.

Regulation of G2/M Progression by the STE Mitogen-activated Protein Kinase Pathway in Budding Yeast Filamentous Growth

Inoculation of diploid budding yeast onto nitrogen-poor agar media stimulates a MAPK pathway to promote filamentous growth. Characteristics of filamentous cells include a specific pattern of gene expression, elongated cell shape, polar budding pattern, persistent attachment to the mother cell, and a distinct cell cycle characterized by cell size control at G2/M. Although a requirement for MAPK signaling in filamentous gene expression is well established, the role of this pathway in the regulation of morphogenesis and the cell cycle remains obscure. We find that ectopic activation of the MAPK signal pathway induces a cell cycle shift to G2/M coordinately with other changes characteristic of filamentous growth. These effects are abrogated by overexpression of the yeast mitotic cyclins Clb1 and Clb2. In turn, yeast deficient for Clb2 or carrying cdc28-1N, an allele of CDK defective for mitotic functions, display enhanced filamentous differentiation and supersensitivity to the MAPK signal. Importantly, activation of Swe1-mediated inhibitory phosphorylation of Thr-18 and/or Tyr-19 of Cdc28 is not required for the MAPK pathway to affect the G2/M delay. Mutants expressing a nonphosphorylatable mutant Cdc28 or deficient for Swe1 exhibit low-nitrogen-dependent filamentous growth and are further induced by an ectopic MAPK signal. We infer that the MAPK pathway promotes filamentous growth by a novel mechanism that inhibits mitotic cyclin/CDK complexes and thereby modulates cell shape, budding pattern, and cell-cell connections.

c-mos and cdc2 Cooperate in the Translational Activation of Fibroblast Growth Factor Receptor-1 during Xenopus Oocyte Maturation

During oocyte maturation in Xenopus, previously quiescent maternal mRNAs are translationally activated at specific times. We hypothesized that the translational recruitment of individual messages is triggered by particular cellular events and investigated the potential for known effectors of the meiotic cell cycle to activate the translation of the FGF receptor-1 (XFGFR) maternal mRNA. We found that both c-mos and cdc2 activate the translation of XFGFR. However, although oocytes matured by injection of recombinant cdc2/cyclin B translate normal levels of XFGFR protein, c-mos depletion reduces the level of XFGFR protein induced by cdc2/cyclin B injection. In oocytes blocked for cdc2 activity, injection of mos RNA induced low levels of XFGFR protein, independent of MAPK activity. Through the use of injected reporter RNAs, we show that the XFGFR 3′ untranslated region inhibitory element is completely derepressed by cdc2 alone. In addition, we identified a new inhibitory element through which both mos and cdc2 activate translation. We found that cdc2 derepresses translation in the absence of polyadenylation, whereas mos requires poly(A) extension to activate XFGFR translation. Our results demonstrate that mos and cdc2, in addition to functioning as key regulators of the meiotic cell cycle, cooperate in the translational activation of a specific maternal mRNA during oocyte maturation.

Two Distinct Signaling Pathways Downstream of Janus Kinase 2 Play Redundant Roles for Antiapoptotic Activity of Granulocyte-Macrophage Colony-stimulating Factor

Human granulocyte-macrophage colony-stimulating factor (hGM-CSF) induces proliferation and sustains the viability of the mouse interleukin-3-dependent cell line BA/F3 expressing the hGM-CSF receptor. Analysis of the antiapoptosis activity of GM-CSF receptor βc mutants showed that box1 but not the C-terminal region containing tyrosine residues is essential for GM-CSF-dependent antiapoptotic activity. Because βc mutants, which activate Janus kinase 2 but neither signal transducer and activator of transcription 5 nor the MAPK cascade sustain antiapoptosis activity, involvement of Janus kinase 2, excluding the above molecules, in antiapoptosis activity seems likely. GM-CSF activates phosphoinositide-3-OH kinase as well as Akt, and activation of both was suppressed by addition of wortmannin. Interestingly, wortmannin did not affect GM-CSF-dependent antiapoptosis, thus indicating that the phosphoinositide-3-OH kinase pathway is not essential for cell surivival. Analysis using the tyrosine kinase inhibitor genistein and a MAPK/extracellular signal-regulated kinase (ERK) kinase 1 inhibitor, PD98059, indicates that activation of either the genistein-sensitive signaling pathway or the PD98059-sensitive signaling pathway from βc may be sufficient to suppress apoptosis. Wild-type and a βc mutant lacking tyrosine residues can induce expression of c-myc and bcl-xL genes; however, drug sensitivities for activation of these genes differ from those for antiapoptosis activity of GM-CSF, which means that these gene products may be involved yet are inadequate to promote cell survival.

Roles for Basal and Stimulated p21Cip-1/WAF1/MDA6 Expression and Mitogen-activated Protein Kinase Signaling in Radiation-induced Cell Cycle Checkpoint Control in Carcinoma Cells

We investigated the role of the cdk inhibitor protein p21Cip-1/WAF1/MDA6 (p21) in the ability of MAPK pathway inhibition to enhance radiation-induced apoptosis in A431 squamous carcinoma cells. In carcinoma cells, ionizing radiation (2 Gy) caused both primary (0–10 min) and secondary (90–240 min) activations of the MAPK pathway. Radiation induced p21 protein expression in A431 cells within 6 h via secondary activation of the MAPK pathway. Within 6 h, radiation weakly enhanced the proportion of cells in G1 that were p21 and MAPK dependent, whereas the elevation of cells present in G2/M at this time was independent of either p21 expression or MAPK inhibition. Inhibition of the MAPK pathway increased the proportion of irradiated cells in G2/M phase 24–48 h after irradiation and enhanced radiation-induced apoptosis. This correlated with elevated Cdc2 tyrosine 15 phosphorylation, decreased Cdc2 activity, and decreased Cdc25C protein levels. Caffeine treatment or removal of MEK1/2 inhibitors from cells 6 h after irradiation reduced the proportion of cells present in G2/M phase at 24 h and abolished the ability of MAPK inhibition to potentiate radiation-induced apoptosis. These data argue that MAPK signaling plays an important role in the progression/release of cells through G2/M phase after radiation exposure and that an impairment of this progression/release enhances radiation-induced apoptosis. Surprisingly, the ability of irradiation/MAPK inhibition to increase the proportion of cells in G2/M at 24 h was found to be dependent on basal p21 expression. Transient inhibition of basal p21 expression increased the control level of apoptosis as well as the abilities of both radiation and MEK1/2 inhibitors to cause apoptosis. In addition, loss of basal p21 expression significantly reduced the capacity of MAPK inhibition to potentiate radiation-induced apoptosis. Collectively, our data argue that MAPK signaling and p21 can regulate cell cycle checkpoint control in carcinoma cells at the G1/S transition shortly after exposure to radiation. In contrast, inhibition of MAPK increases the proportion of irradiated cells in G2/M, and basal expression of p21 is required to maintain this effect. Our data suggest that basal and radiation-stimulated p21 may play different roles in regulating cell cycle progression that affect cell survival after radiation exposure.

MEK kinase 1 is critically required for c-Jun N-terminal kinase activation by proinflammatory stimuli and growth factor-induced cell migration

Exposure of eukaryotic cells to extracellular stimuli results in activation of mitogen-activated protein kinase (MAPK) cascades composed of MAPKs, MAPK kinases (MAP2Ks), and MAPK kinase kinases (MAP3Ks). Mammals possess a large number of MAP3Ks, many of which can activate the c-Jun N-terminal kinase (JNK) MAPK cascade when overexpressed, but whose biological function is poorly understood. We examined the function of the MAP3K MEK kinase 1 (MEKK1) in proinflammatory signaling. Using MEKK1-deficient embryonic stem cells prepared by gene targeting, we find that, in addition to its function in JNK activation by growth factors, MEKK1 is required for JNK activation by diverse proinflammatory stimuli, including tumor necrosis factor α, IL-1, double-stranded RNA, and lipopolysaccharide. MEKK1 is also essential for induction of embryonic stem cell migration by serum factors, but is not required for activation of other MAPKs or the IκB kinase signaling cascade.

Role of endocytosis in the activation of the extracellular signal-regulated kinase cascade by sequestering and nonsequestering G protein-coupled receptors

Acting through a number of distinct pathways, many G protein-coupled receptors (GPCRs) activate the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) cascade. Recently, it has been shown that in some cases, clathrin-mediated endocytosis is required for GPCR activation of the ERK/MAPK cascade, whereas in others it is not. Accordingly, we compared ERK activation mediated by a GPCR that does not undergo agonist-stimulated endocytosis, the α2A adrenergic receptor (α2A AR), with ERK activation mediated by the β2 adrenergic receptor (β2 AR), which is endocytosed. Surprisingly, we found that in COS-7 cells, ERK activation by the α2A AR, like that mediated by both the β2 AR and the epidermal growth factor receptor (EGFR), is sensitive to mechanistically distinct inhibitors of clathrin-mediated endocytosis, including monodansylcadaverine, a mutant dynamin I, and a mutant β-arrestin 1. Moreover, we determined that, as has been shown for many other GPCRs, both α2A and β2 AR-mediated ERK activation involves transactivation of the EGFR. Using confocal immunofluorescence microscopy, we found that stimulation of the β2 AR, the α2A AR, or the EGFR each results in internalization of a green fluorescent protein-tagged EGFR. Although β2 AR stimulation leads to redistribution of both the β2 AR and EGFR, activation of the α2A AR leads to redistribution of the EGFR but the α2A AR remains on the plasma membrane. These findings separate GPCR endocytosis from the requirement for clathrin-mediated endocytosis in EGFR transactivation-mediated ERK activation and suggest that it is the receptor tyrosine kinase or another downstream effector that must engage the endocytic machinery.

Survival function of ERK1/2 as IL-3-activated, staurosporine-resistant Bcl2 kinases

Bcl2 phosphorylation at Ser-70 may be required for the full and potent suppression of apoptosis in IL-3-dependent myeloid cells and can result from agonist activation of mitochondrial protein kinase C (PKC). Paradoxically, expression of exogenous Bcl2 can protect parental cells from apoptosis induced by the potent PKC inhibitor, staurosporine (stauro). High concentrations of stauro of up to 1 μM only partially inhibit IL-3-stimulated Bcl2 phosphorylation but completely block PKC-mediated Bcl2 phosphorylation in vitro. These data indicate a role for a stauro-resistant Bcl2 kinase (SRK). We show that aurintricarboxylic acid (ATA), a nonpeptide activator of cellular MEK/mitogen-activated protein kinase (MAPK) kinase, can induce Ser-70 phosphorylation of Bcl2 and support survival of cells expressing wild-type but not the phosphorylation-incompetent S70A mutant Bcl2. A role for a MEK/MAPK as a responsible SRK was implicated because the highly specific MEK/MAPK inhibitor, PD98059, also can only partially inhibit IL-3-induced Bcl2 phosphorylation, whereas the combination of PD98059 and stauro completely blocks phosphorylation and synergistically enhances apoptosis. p44MAPK/extracellular signal-regulated kinase 1 (ERK1) and p42 MAPK/ERK2 are activated by IL-3, colocalize with mitochondrial Bcl2, and can directly phosphorylate Bcl2 on Ser-70 in a stauro-resistant manner both in vitro and in vivo. These findings suggest a role for the ERK1/2 kinases as SRKs. Thus, the SRKs can serve to functionally link the IL-3-stimulated proliferative and survival signaling pathways and, in a novel capacity, may explain how Bcl2 can suppress stauro-induced apoptosis. In addition, although the mechanism of regulation of Bcl2 by phosphorylation is not yet clear, our results indicate that phosphorylation may functionally stabilize the Bcl2-Bax heterodimerization.

Neuropeptide Y (NPY) potentiates phenylephrine-induced mitogen-activated protein kinase activation in primary cardiomyocytes via NPY Y5 receptors

Neuropeptide Y (NPY) has been shown to participate in the cardiovascular response mediated by the sympathetic system. In this report, we investigate the growth factor properties of NPY on cardiac myocytes. Mitogen-activated protein kinases (MAPK) are key signaling molecules in the transduction of trophic signals. Therefore, the role of NPY in inducing MAPK activation was studied in mouse neonatal cardiomyocytes. Exposure of neonatal cardiomyocytes to either NPY, phenylephrine, or angiotensin II induces a rapid phosphorylation of the extracellular responsive kinase, the c-jun N-terminal kinase, and the p38 kinase as well as an activation of protein kinase C (PKC). Moreover, NPY potentiates phenylephrine-induced MAPK and PKC stimulation. In contrast, NPY has no synergistic effect on angiotensin II-stimulated MAPK phosphorylation or PKC activity. NPY effects are pertussis toxin-sensitive and calcium-independent and are mediated by NPY Y5 receptors. Taken together, these results suggest that NPY, via Gi protein-coupled NPY Y5 receptors, could participate in the development of cardiac hypertrophy during chronic sympathetic stimulation by potentiating α-adrenergic signals.

Endogenous, hyperactive Rac3 controls proliferation of breast cancer cells by a p21-activated kinase-dependent pathway

Uncontrolled cell proliferation is a major feature of cancer. Experimental cellular models have implicated some members of the Rho GTPase family in this process. However, direct evidence for active Rho GTPases in tumors or cancer cell lines has never been provided. In this paper, we show that endogenous, hyperactive Rac3 is present in highly proliferative human breast cancer-derived cell lines and tumor tissues. Rac3 activity results from both its distinct subcellular localization at the membrane and altered regulatory factors affecting the guanine nucleotide state of Rac3. Associated with active Rac3 was deregulated, persistent kinase activity of two isoforms of the Rac effector p21-activated kinase (Pak) and of c-Jun N-terminal kinase (JNK). Introducing dominant-negative Rac3 and Pak1 fragments into a breast cancer cell line revealed that active Rac3 drives Pak and JNK kinase activities by two separate pathways. Only the Rac3–Pak pathway was critical for DNA synthesis, independently of JNK. These findings identify Rac3 as a consistently active Rho GTPase in human cancer cells and suggest an important role for Rac3 and Pak in tumor growth.

Sustained activation of Ras/Raf/mitogen-activated protein kinase cascade by the tumor suppressor p53

The p53 tumor suppressor gene can inhibit proliferation transiently, induce permanent cell-cycle arrest/senescence, or cause apoptosis depending on the cellular context. The mitogen-activated protein kinase (MAPK) cascade is known to play a crucial role in cell proliferation and differentiation. Moreover, the duration and intensity of MAPK activation can profoundly influence the biological response observed. We demonstrated that a sustained activation of MAPK cascade could be induced by wild-type p53 expression but not by p21Waf1/Cip1. Furthermore, exposure of normal cells to DNA-damaging agents induced MAPK activation in a p53-dependent manner. Tumor-derived p53 mutants defective in DNA binding failed to activate MAPK, implying that p53 transcriptional activity is essential for this function. Finally, activation of MAPK by p53 was inhibited by expression of dominant-negative Ras (N17Ras) and Raf1 mutants, indicating that MAPK activation by p53 is mediated at a level upstream of Ras. All of these findings establish a biochemical link between p53 signaling and the Ras/Raf/MAPK cascade.