Adaptor proteins Grb2 and Crk couple Pyk2 with activation of specific mitogen-activated protein kinase cascades.

The protein tyrosine kinase Pyk2 acts as an upstream regulator of mitogen-activated protein (MAP) kinase cascades in response to numerous extracellular signals. The precise molecular mechanisms by which Pyk2 activates distinct MAP kinase pathways are not yet fully understood. In this report, we provide evidence that the protein tyrosine kinase Src and adaptor proteins Grb2, Crk, and p130Cas act as downstream mediators of Pyk2 leading to the activation of extracellular signal-regulated kinase (ERK) and c-Jun amino-terminal kinase (JNK). Pyk2-induced activation of Src is necessary for phosphorylation of Shc and p130Cas and their association with Grb2 and Crk, respectively, and for the activation of ERK and JNK cascades. Expression of a Grb2 mutant with a deletion of the amino-terminal Src homology 3 domain or the carboxyl-terminal tail of Sos strongly reduced Pyk2-induced ERK activation, with no apparent effect on JNK activity. Grb2 with a deleted carboxyl-terminal Src homology 3 domain partially blocked Pyk2-induced ERK and JNK pathways, whereas expression of dominant interfering mutants of p130Cas or Crk specifically inhibited JNK but not ERK activation by Pyk2. Taken together, our data reveal specific pathways that couple Pyk2 with MAP kinases: the Grb2/Sos complex connects Pyk2 to the activation of ERK, whereas adaptor proteins p130Cas and Crk link Pyk2 with the JNK pathway.

Cell adhesion regulates the interaction between the docking protein p130(Cas) and the 14-3-3 proteins.

Integrin ligand binding induces a signaling complex formation via the direct association of the docking protein p130(Cas) (Cas) with diverse molecules. We report here that the 14-3-3zeta protein interacts with Cas in the yeast two-hybrid assay. We also found that the two proteins associate in mammalian cells and that this interaction takes place in a phosphoserine-dependent manner, because treatment of Cas with a serine phosphatase greatly reduced its ability to bind 14-3-3zeta. Furthermore, the Cas-14-3-3zeta interaction was found to be regulated by integrin-mediated cell adhesion. Thus, when cells are detached from the extracellular matrix, the binding of Cas to 14-3-3zeta is greatly diminished, whereas replating the cells onto fibronectin rapidly induces the association. Consistent with these results, we found that the subcellular localization of Cas and 14-3-3 is also regulated by integrin ligand binding and that the two proteins display a significant co-localization during cell attachment to the extracellular matrix. In conclusion, our results demonstrate that 14-3-3 proteins participate in integrin-activated signaling pathways through their interaction with Cas, which, in turn, may contribute to important biological responses regulated by cell adhesion to the extracellular matrix.

Met-induced JNK activation is mediated by the adapter protein Crk and correlates with the Gab1 - Crk signaling complex formation.

Constitutive activation of the Met tyrosine kinase results in transformation of cells of diverse origin. Recent studies have demonstrated a role for the c-Jun N-terminal kinase (JNK) in Met-induced transformation, but little is known about the molecular mechanisms that connect Met to JNK activation. Our studies show that activated Met associates with, and phosphorylates, the docking protein Gab1, which in turn binds to the src homology 2 (SH2)-domain of the adapter protein Crk and recruits Crk to the Met signaling complex. Formation of the Gab1 - Crk complex correlates with Met-induced JNK activation, and mutant forms of Met that fail to induce the complex formation also fail to activate JNK. Importantly, expression of a loss-of-function mutant of Crk severely impairs activation of the JNK pathway by Met. We also show here that Met controls the transcription of the matrix metalloproteinase-1 (MMP-1) gene in carcinoma cells and that this transcriptional regulation occurs in a Crk - JNK-dependent manner through an AP-1 element in the MMP-1 promoter. Taken together, our data implicate the Gab1 - Crk signaling complex in Met-induced JNK activation and suggest that the Gab1 - Crk complex formation may be an important event in regulating the tumorigenic phenotype of Met-transformed cells.

The adaptor protein Crk connects multiple cellular stimuli to the JNK signaling pathway.

c-Jun N-terminal kinases (JNKs) are potently activated by a number of cellular stimuli. Small GTPases, in particular Rac, are responsible for initiating the activation of the JNK pathways. So far, the signals leading from extracellular stimuli to the activation of Rac have remained elusive. Recent studies have demonstrated that the Src homology 2 (SH2)- and Src homology 3 (SH3)-containing adaptor protein Crk is capable of activating JNK when ectopically expressed. We found here that transient expression of Crk induces JNK activation, and this activation was dependent on both the SH2- and SH3-domains of Crk. Expression of p130(Cas) (Cas), a major binding protein for the Crk SH2-domain, also induced JNK activation, which was blocked by the SH2-mutant of Crk. JNK activation by Cas and Crk was effectively blocked by a dominant-negative form of Rac, suggesting for a linear pathway from the Cas-Crk-complex to the Rac-JNK activation. Many of the stimuli that activate the Rac-JNK pathway enhance engagement of the Crk SH2-domain. JNK activation by these stimuli, such as epidermal growth factor, integrin ligand binding and v-Src, was efficiently blocked by dominant-negative mutants of Crk. A dominant-negative form of Cas in turn blocked the integrin-, but not epidermal growth factor - nor v-Src-mediated JNK activation. Together, these results demonstrate an important role for Crk in connecting multiple cellular stimuli to the Rac-JNK pathway, and a role for the Cas-Crk complex in integrin-mediated JNK activation.

Extracellular signal-regulated kinase and c-Jun NH2-terminal kinase activation by mechanical stretch is integrin-dependent and matrix-specific in rat cardiac fibroblasts.

Integrins, which connect the cytoskeleton to the extracellular matrix and mediate a variety of signaling cascades, may transduce mechanical stimuli into biochemical signals. We studied integrin- and matrix-dependent activation of extracellular signal-regulated kinase (ERK2), c-Jun NH2-terminal kinase (JNK1), and p38 in response to 4% static biaxial stretch in rat cardiac fibroblasts. ERK2 and JNK1, but not p38, were rapidly activated by stretch when the fibroblasts were allowed to synthesize their own matrices. When the cells were limited to specific matrix substrates, ERK2 and JNK1 were differentially activated: ERK2 was only activated when the cells were plated on fibronectin, while JNK1 was activated when the cells were plated on fibronectin, vitronectin, or laminin. Plating cells on collagen before stretching did not activate either kinase. Adhesion to all matrices was integrin-dependent because it could be blocked by inhibitors of specific integrins. ERK2 activation could be blocked with a combination of anti-alpha4 and -alpha5 antibodies and an arginine-glycine-aspartic acid (RGD) peptide, while the antibodies or peptide used separately failed to block ERK2 activation. This result suggests that at least two integrins, alpha4beta1 and an RGD-directed, non-alpha5beta1 integrin, activate ERK2 in response to mechanical stimulation. Activation of JNK1 could not be blocked with the inhibitors, suggesting that an RGD-independent integrin or integrins other than alpha4beta1 can activate JNK1 in cells adherent to fibronectin. This study demonstrates that integrins act as mechanotransducers, providing insight into potential mechanisms for in vivo responses to mechanical stimuli.

The proto-oncogene product p120(cbl) links c-Src and phosphatidylinositol 3'-kinase to the integrin signaling pathway.

Integrin-mediated cell adhesion triggers intracellular signaling cascades, including tyrosine phosphorylation of intracellular proteins. We show in this report that p120(cbl) (Cbl), the 120-kDa c-cbl proto-oncogene product, becomes tyrosine-phosphorylated during integrin-mediated macrophage cell adhesion to extracellular matrix substrata and anti-integrin antibodies. This tyrosine phosphorylation does not occur when cells attach to polylysine, to which cells adhere in a nonspecific fashion. It also does not take place when adhesion-induced reorganization of the cytoskeleton is inhibited with cytochalasin D. In contrast to the rapid and transient tyrosine phosphorylation of Cbl by CSF-1 stimulation, tyrosine phosphorylation of Cbl by cell attachment was gradual and persistent. Tyrosine-phosphorylated Cbl was found to form complexes with the SH2 domain-containing signaling proteins Src and phosphatidylinositol 3-kinase; in vitro kinase assays demonstrated that these kinases were active in the Cbl complexes following integrin ligand binding. Furthermore, Cbl was found to translocate to the plasma membrane in response to cell adhesion to fibronectin. These observations suggest that Cbl serves as a docking protein and may transduce signals to downstream signaling pathways following integrin-mediated cell adhesion in macrophages.

Induction of c-fos expression through JNK-mediated TCF/Elk-1 phosphorylation.

Growth factors induce c-fos transcription by stimulating phosphorylation of transcription factor TCF/Elk-1, which binds to the serum response element (SRE). Under such conditions Elk-1 could be phosphorylated by the mitogen-activated protein kinases (MAPKs) ERK1 and ERK2. However, c-fos transcription and SRE activity are also induced by stimuli, such as UV irradiation and activation of the protein kinase MEKK1, that cause only an insignificant increase in ERK1/2 activity. However, both of these stimuli strongly activate two other MAPKs, JNK1 and JNK2, and stimulate Elk-1 transcriptional activity and phosphorylation. We find that the JNKs are the predominant Elk-1 activation domain kinases in extracts of UV-irradiated cells and that immunopurified JNK1/2 phosphorylate Elk-1 on the same major sites recognized by ERK1/2, that potentiate its transcriptional activity. Finally, we show that UV irradiation, but not serum or phorbol esters, stimulate translocation of JNK1 to the nucleus. As Elk-1 is most likely phosphorylated while bound to the c-fos promoter, these results suggest that UV irradiation and MEKK1 activation stimulate TCF/Elk-1 activity through JNK activation, while growth factors induce c-fos through ERK activation.

PDGF receptor as a specific in vivo target for low M(r) phosphotyrosine protein phosphatase.

Low M(r) phosphotyrosine protein phosphatase (LMW-PTP) is a 18 kDa cytosolic enzyme widely distributed in eukaryotic cells. LMW-PTP catalyses the hydrolysis of phosphotyrosine residues and overexpression of the enzyme in normal and transformed cells inhibits cell proliferation. Site directed mutagenesis, together with crystallographic studies, have contributed to clarify the catalytic mechanism, which involves the active site signature sequence C12XXXXXR18, a main feature of all PTPase family members. In order to identify the LMW-PTP substrate/s we have expressed in NIH-3T3 cells a catalytically inert Cys12 to Ser phosphatase mutant which has preserved its capacity for substrate binding. Overexpression of the mutant phosphatase leads to enhanced cell proliferation and serum induced mitogenesis, indicating that the mutation results in the production of a dominant negative protein. Analysis of mutant LMW-PTP expressing cells has enabled us to demonstrate an association between LMW-PTP and platelet derived growth factor receptor that appears to be highly specific. Our data suggest a catalytic action of LMW-PTP on the phosphorylated platelet derived growth factor receptor.

ras-p21 activates phospholipase D and A2, but not phospholipase C or PKC, in Xenopus laevis oocytes.

Xenopus laevis oocytes are a powerful tool for the characterization of signal transduction pathways leading to the induction of DNA synthesis. Since activation of PLA2, PLC, or PLD has been postulated as a mediator of ras function, we have used the oocyte system to study the putative functional relationship between ras-p21 and these phospholipases. A rapid generation of PA and DAG was observed after ras-p21 microinjection, suggesting the activation of both PLC and PLD enzymes. However, production of DAG was sensitive to inhibition of the PA-hydrolase by propranolol, indicating that PLD is the enzyme responsible for the generation of both PA and DAG. Microinjection of PLD or ras-p21 induced the late production of lysophosphatidylcholine on a p42MAPK-dependent manner, an indication of the activation of a PLA2. Inhibition of this enzyme by quinacrine does not inhibit PLD- or ras-induced GVBD, suggesting that PLA2 activation is not needed for ras or PLD function. Contrary to 3T3 fibroblasts, where ras-p21 is functionally dependent for its mitogenic activity on TPA- and staurosporine-sensitive PKC isoforms, in Xenopus oocytes, induction of GVBD by ras-p21 was independent of PKC, while PLC-induced GVBD was sensitive to PKC inhibition. Thus, our results demonstrate the activation of PLD and PLA2 by ras-p21 proteins, while no effect on PLC was observed.

[Acute amanita phalloides poisoning in the second pregnancy trimester]. / Intossicazione acuta da amanita phalloide nel secondo trimestre di gravidanza.

A 26-year old woman who suffered from mushroom poisoning due to amanita phalloides at 22 weeks gestation. When the life threatening maternal illness was overcome gestation went on until full-term, natural birth. The child was born in perfect physical conditions, without noticeable symptoms of hepatic injury.

Phosphorylcholine: a novel second messenger essential for mitogenic activity of growth factors.

Growth factor stimulation of quiescent cells induces a series of intracellular early and late events that ultimately lead to DNA synthesis and cell division. We describe here that production of phosphorylcholine is an essential component of the late events involved in the induction of DNA synthesis by platelet-derived growth factor (PDGF-BB), a prototype mitogen for fibroblasts. Moreover, phosphorylcholine itself is mitogenic when added exogenously to NIH3T3 cells, further indicating its role as a crucial intracellular messenger for DNA synthesis. Choline kinase, the first step in the route of phosphatidylcholine synthesis appears to be the critical regulatory enzyme in phosphorylcholine production, indicating that regulation of choline kinase represents a key step during mitogenic stimulation. We also describe that several growth factors (PDGF-AA, basic FGF, EGF and phorbol esters) rely on their ability to generate phosphorylcholine for their proliferating activity. In contrast, DNA synthesis induced by serum did not require phosphorylcholine. Moreover, the requirement for phosphorylcholine production in PDGF-stimulated cells can be over-ruled by addition of insulin. Thus, cell proliferation in NIH3T3 cells can be triggered off by alternative pathways and one of them involves generation of phosphorylcholine.

Acylphosphatase synergizes with progesterone during maturation of Xenopus laevis oocytes.

Xenopus laevis oocytes are physiologically arrested in the G2/M phase border of the first meiotic division. A number of different stimuli can trigger off the re-entry into the cell cycle as a consequence of activation of either membrane-dependent or -independent intracellular signals. This system has been widely used to study signal transduction mechanisms induced by hormones. Among those more intensively researched, special attention has been devoted to elucidate the mechanism of activation induced by progesterone. However, despite intense efforts to understand the intracellular signalling mechanism of progesterone, a clear notion of the most relevant events involved in this process has not yet been elucidated. We provide evidence that acylphosphatase, an enzyme responsible for the regulation of membrane pumps in eukaryotic cells, synergizes with progesterone for induction of oocyte maturation. We deduced that this synergism may be related to the regulation of intracellular Ca2+ levels for several reasons: (1) maturation of oocytes by extracellular Ca2+ is blocked by acylphosphatase; (2) both progesterone and acylphosphatase drastically reduced Ca2+ uptake; (3) progesterone-induced maturation does not depend on a rise in intracellular Ca2+, since microinjection of EGTA, a calcium chelator, does not affect maturation induced by progesterone.

Dephosphorylation of tyrosine phosphorylated synthetic peptides by rat liver phosphotyrosine protein phosphatase isoenzymes.

Five phosphotyrosine-containing peptides have been synthesized by FMOC solid-phase peptide synthesis. These peptides correspond to the 411-419 sequence of the Xenopus src oncogene, to the 1191-1220 sequence of the human EGF receptor precursor, to the 1146-1158 sequence of the human insulin receptor, to the 856-865 sequence of the human beta-PDGF receptor, and to the 5-16 sequence of the erythrocyte human band 3. The peptides were used as substrates for activity assay of two isoforms (AcP1 and AcP2) of a low molecular weight cytosolic PTPase. The assay, performed in microtiter EIA plates using Malachite green to determine the released phosphate, was rapid, reproducible, and sensitive. Both PTPase isoforms were able to hydrolyze all synthesized peptides, though with different affinity and rate. The main kinetic parameters were compared and discussed with respect to the role of the two enzymes in the cell.

Microinjection of acylphosphatase blocks Xenopus laevis oocytes maturation induced by ras-p21.

Ras proteins induce germinal vesicle breakdown (GVBD) when microinjected into Xenopus laevis oocytes. The mechanism of action is still unresolved, although several hypotheses have been proposed. Acylphosphatase is a cytosolic enzyme that specifically catalyses the hydrolysis of the carboxylphosphate bond of acylphosphate for the removal of acylphosphate residues of various membrane pumps. A direct effect of acylphosphatase on the regulation of ionic balance of a cell by interaction with ionic membrane pumps has been proposed. We have analyzed the effect of microinjecting acylphosphatase, by itself or along with ras-p21 proteins or progesterone, into oocytes. The enzyme alone is unable to induce GVBD, but increases oocyte maturation induced by progesterone. By contrast, acylphosphatase blocked GVBD induced by microinjection of oncogenic ras-p21. These data suggest that acylphosphatase acts synergistically or antagonistically with factors involved in proliferating signals by altering the intracellular ionic conditions of the cell, conforming the hypothesis that the intracellular ionic condition of the cell is important in the induction of proliferating signals, and that its perturbation may have a serious effect on signal transduction.