Activation of Mitogen-Activated Protein Kinase in Cultured Rat Hippocampal Neurons by Stimulation of Glutamate Receptors

Abstract: Mitogen-activated protein kinase (MAP kinase) was activated by stimulation of glutamate receptors in cultured rat hippocampal neurons. Ten micromolar glutamate maximally stimulated MAP kinase activity, which peaked during 10 min and decreased to the basal level within 30 min. Experiments using glutamate receptor agonists and antagonists revealed that glutamate stimulated MAP kinase through NMDA and metabotropic glutamate receptors but not through non-NMDA receptors. Glutamate and its receptor agonists had no apparent effect on MAP kinase activation in cultured cortical astrocytes. Addition of calphostin C, a protein kinase C (PKC) inhibitor, or down-regulation of PKC activity partly abolished the stimulatory effect by glutamate, but the MAP kinase activation by treatment with ionomycin, a Ca²⁺ ionophore, remained intact. Lavendustin A, a tyrosine kinase inhibitor, was without effect. In experiments with ³²P-labeled hippocampal neurons, MAP kinase activation by glutamate was associated with phosphorylation of the tyrosine residue located on MAP kinase. However, phosphorylation of Raf-1, the c- raf protooncogene product, was not stimulated by treatment with glutamate. Our observations suggest that MAP kinase activation through glutamate receptors in hippocampal neurons is mediated by both the PKC-dependent and the Ca²⁺-dependent pathways and that the activation of Raf-1 is not involved.     

B-Raf-dependent regulation of the MEK-1/mitogen-activated protein kinase pathway in PC12 cells and regulation by cyclic AMP.

Growth factor receptor tyrosine kinase regulation of the sequential phosphorylation reactions leading to mitogen-activated protein (MAP) kinase activation in PC12 cells has been investigated. In response to epidermal growth factor, nerve growth factor, and platelet-derived growth factor, B-Raf and Raf-1 are activated, phosphorylate recombinant kinase-inactive MEK-1, and activate wild-type MEK-1. MEK-1 is the dual-specificity protein kinase that selectively phosphorylates MAP kinase on tyrosine and threonine, resulting in MAP kinase activation. B-Raf and Raf-1 are growth factor-regulated Raf family members which regulate MEK-1 and MAP kinase activity in PC12 cells. Protein kinase A activation in response to elevated cyclic AMP (cAMP) levels inhibited B-Raf and Raf-1 stimulation in response to growth factors. Ras.GTP loading in response to epidermal growth factor, nerve growth factor, or platelet-derived growth factor was unaffected by protein kinase A activation. Even though elevated cAMP levels inhibited Raf activation, the growth factor activation of MEK-1 and MAP kinase was unaffected in PC12 cells. The results demonstrate that tyrosine kinase receptor activation of MEK-1 and MAP kinase in PC12 cells is regulated by B-Raf and Raf-1, whose activation is inhibited by protein kinase A, and MEK activators, whose activation is independent of cAMP regulation.     

Differential regulation of Raf-1 and B-Raf and Ras-dependent activation of mitogen-activated protein kinase by cyclic AMP in PC12 cells.

Growth factor stimulation of the mitogen-activated protein (MAP) kinase pathway in fibroblasts is inhibited by cyclic AMP (cAMP) as a result of inhibition of Raf-1. In contrast, cAMP inhibits neither nerve growth factor-induced MAP kinase activation nor differentiation in PC12 pheochromocytoma cells. Instead, in PC12 cells cAMP activates MAP kinase. Since one of the major differences between the Ras/Raf/MAP kinase cascades of these cell types is the expression of B-Raf in PC12 cells, we compared the effects of cAMP on Raf-1 and B-Raf. In PC12 cells maintained in serum-containing medium, B-Raf was refractory to inhibition by cAMP, whereas Raf-1 was effectively inhibited. In contrast, both B-Raf and Raf-1 were inhibited by cAMP in serum-starved PC12 cells. The effect of cAMP is thus dependent upon growth conditions, with B-Raf being resistant to cAMP inhibition in the presence of serum. These results were extended by studies of Rat-1 fibroblasts into which B-Raf had been introduced by transfection. As in PC12 cells, B-Raf was resistant to inhibition by cAMP in the presence of serum, whereas Raf-1 was effectively inhibited. In addition, the expression of B-Raf rendered Rat-1 cells resistant to the inhibitory effects of cAMP on both growth factor-induced activation of MAP kinase and mitogenesis. These results indicate that Raf-1 and B-Raf are differentially sensitive to inhibition by cAMP and that B-Raf expression can contribute to cell type-specific differences in the regulation of the MAP kinase pathway. In contrast to the situation in PC12 cells, cAMP by itself did not stimulate MAP kinase in B-Raf-expressing Rat-1 cells. The activation of MAP kinase by cAMP in PC12 cells was inhibited by the expression of a dominant negative Ras mutant, indicating that cAMP acts on a target upstream of Ras. Thus, it appears that a signaling component upstream of Ras is also require for cAMP stimulation of MAP kinase in PC12 cells.     

Picrosides I and II,selective enhancers of the mitogen-activated protein kinase-dependent signaling pathway in the action of neuritogenic substances on PC12D cells

Picrosides I and II caused a concentration-dependent (> 0.1 microM) enhancement of basic fibroblast growth factor (bFGF, 2 ng/ml)-, staurosporine (10 nM)- and dibutyryl cyclic AMP (dbcAMP, 0.3 mM)-induced neurite outgrowth from PC12D cells. PD98059 (20 microM), a potent mitogen-activated protein (MAP) kinase kinase inhibitor, blocked the enhancement of bFGF (2 ng/ml)-, staurosporine (10 nM)- or dbcAMP (0.3 mM)-induced neurite outgrowth by picrosides, suggesting that picrosides activate MAP kinase-dependent signaling pathway. However, PD98059 did not affect the bFGF (2 ng/ml)-, staurosporine (10 nM)- and dbcAMP (0.3 mM)-induced neurite outgrowth in PC12D cells, indicating the existence of two components in neurite outgrowth induced by bFGF, staurosporine and dbcAMP, namely the MAP kinase-independent and the masked MAP kinase-dependent one. Furthermore, picrosides-induced enhancements of the bFGF-action were markedly inhibited by GF109203X (0.1 microM), a protein kinase C inhibitor. The expression of phosphorylated MAP kinase was markedly increased by bFGF (2 ng/ml) and dbcAMP (0.3 mM), whereas that was not enhanced by staurosporine (10 nM). Picrosides had no effect on the phosphorylation of MAP kinase induced by bFGF or dbcAMP and also unaffected it in the presence of staurosporine. These results suggest that picrosides I and II enhance bFGF-, staurosporine- or dbcAMP-induced neurite outgrowth from PC12D cells, probably by amplifying a down-stream step of MAP kinase in the intracellular MAP kinase-dependent signaling pathway. Picrosides I and II may become selective pharmacological tools for studying the MAP kinase-dependent signaling pathway in outgrowth of neurites induced by many kinds of neuritogenic substances including bFGF.     

Angiotensin III Induces c-Jun N-terminal Kinase Leading to Proliferation of Rat Astrocytes

Previously, we showed in cultured rat astrocytes that angiotensin (Ang) III induced astrocyte proliferation and phosphorylation of ERK1/2 mitogen activated protein (MAP) kinases through interaction with the AT(1) receptor. In the current study, we determined whether the c-Jun N terminal kinase (JNK) MAP kinase pathway was similarly affected by the peptide in cultured brainstem astrocytes. Ang III induced JNK phosphorylation in a concentration- and time-dependent manner. Similar to ERK1/2 phosphorylation, maximal phosphorylation occurred with 100 nM Ang III and was apparent within a minute of exposure to the peptide. Peak effects were observed over a 5-15 min time range. Pretreatment of brainstem astrocytes with the JNK inhibitor, SP600125, prevented Ang III phosphorylation of JNK, as well as Ang III-mediated astrocyte growth. The selective AT(1) receptor antagonist, Losartan, prevented Ang III-induced JNK phosphorylation. Pretreatment of astrocytes with the AT(2) receptor blocker PD123319 was ineffective in preventing JNK phosphorylation by Ang III. Interestingly, both Ang II and Ang III induced JNK phosphorylation to a similar extent suggesting that the two peptides were equipotent in this effect. Our findings suggest that Ang III interacts with Ang AT(1) receptors to directly stimulate the JNK MAP kinase pathway leading to astrocyte growth. This study is the first to show that Ang III actions may involve the JNK MAP kinase pathway in astrocytes and provide key information that may lead to a better understanding of the functions of Ang III in the central nervous system, in particular in astrocytes.     

cAMP-dependent protein kinase inhibits the mitogenic action of vascular endothelial growth factor and fibroblast growth factor in capillary endothelial cells by blocking Raf activation

Proliferation of endothelial cells is regulated by angiogenic and antiangiogenic factors whose actions are mediated by complex interactions of multiple signaling pathways. Both vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) stimulate cell proliferation and activate the mitogen-activated protein kinase (MAPK) cascade in bovine brain capillary endothelial (BBE) cells. We have extended these findings to show that both mitogens activate MAPK via stimulation of Raf-1. Activation of Raf/MAPK is inhibited by increasing intracellular cAMP levels pharmacologically or via stimulation of endogenously expressed β-adrenergic receptors. Both VEGF- and bFGF-induced Raf-1 activity are blocked in the presence of forskolin or 8-bromo-cAMP by 80%. The actions of increased cAMP appear to be mediated by cAMP-dependent protein kinase (PKA), since treatment with H-89, a the specific inhibitor of PKA, reversed the inhibitory effect of elevated cAMP levels on mitogen-induced cell proliferation and Raf/MAPK activation. Moreover, elevations in cAMP/PKA activity inhibit mitogen-induced cell proliferation. These findings demonstrate, in cultured endothelial cells, that the cAMP/PKA signaling pathway is potentially an important physiological inhibitor of mitogen activation of the MAPK cascade and cell proliferation. J. Cell. Biochem. 67:353–366, 1997. © 1997 Wiley-Liss, Inc.     

p42/p44 MAP kinase activation is localized to caveolae-free membrane domains in airway smooth muscle

Caveolae are abundant plasma membrane invaginations in airway smooth muscle that may function as preorganized signalosomes by sequestering and regulating proteins that control cell proliferation, including receptor tyrosine kinases (RTKs) and their signaling effectors. We previously demonstrated, however, that p42/p44 MAP kinase, a critical effector for cell proliferation, does not colocalize with RTKs in caveolae of quiescent airway myocytes. Therefore, we investigated the subcellular sites of growth factor-induced MAP kinase activation. In quiescent myocytes, though epidermal growth factor receptor (EGFR) was almost exclusively found in caveolae, p42/p44 MAP kinase, Grb2, and Raf-1 were absent from these membrane domains. EGF induced concomitant phosphorylation of caveolin-1 and p42/p44 MAP kinase; however, EGF did not promote the localization of p42/p44 MAP kinase, Grb2, or Raf-1 to caveolae. Interestingly, stimulation of muscarinic M(2) and M(3) receptors that were enriched in caveolae-deficient membranes also induced p42/p44 MAP kinase phosphorylation, but this occurred in the absence of caveolin-1 phosphorylation. This suggests that the localization of receptors to caveolae and interaction with caveolin-1 is not directly required for p42/p44 MAP kinase phosphorylation. Furthermore, we found that EGF exposure induced rapid translocation of EGFR from caveolae to caveolae-free membranes. EGFR trafficking coincided temporally with EGFR and p42/p44 MAP kinase phosphorylation. Collectively, this indicates that although caveolae sequester some receptors associated with p42/p44 MAP kinase activation, the site of its activation is associated with caveolae-free membrane domains. This reveals that directed trafficking of plasma membrane EGFR is an essential element of signal transduction leading to p42/p44 MAP kinase activation.     

Activation of Raf-1 signaling by protein kinase C through a mechanism involving Raf kinase inhibitory protein

Protein kinase C (PKC) regulates activation of the Raf-1 signaling cascade by growth factors, but the mechanism by which this occurs has not been elucidated. Here we report that one mechanism involves dissociation of Raf kinase inhibitory protein (RKIP) from Raf-1. Classic and atypical but not novel PKC isoforms phosphorylate RKIP at serine 153 (Ser-153). RKIP Ser-153 phosphorylation by PKC either in vitro or in response to 12-O-tetradecanoylphorbol-13-acetate or epidermal growth factor causes release of RKIP from Raf-1, whereas mutant RKIP (S153V or S153E) remains bound. Increased expression of PKC can rescue inhibition of the mitogen-activated protein (MAP) kinase signaling cascade by wild-type but not mutant S153V RKIP. Taken together, these results constitute the first model showing how phosphorylation by PKC relieves a key inhibitor of the Raf/MAP kinase signaling cascade and may represent a general mechanism for the regulation of MAP kinase pathways.     

Raf kinase inhibitory protein regulates Raf-1 but not B-Raf kinase activation

Raf kinase inhibitory protein (RKIP; also known as phosphatidylethanolamine-binding protein or PEBP) is a modulator of the Raf/MAPK signaling cascade and a suppressor of metastatic cancer. Here, we show that RKIP inhibits MAPK by regulating Raf-1 activation; specifically, RKIP acts subsequent to Raf-1 membrane recruitment, prevents association of Raf-1 and p21-activated kinase (PAK), and blocks phosphorylation of the Raf-1 kinase domain by PAK and Src family kinases. Mutation of the PAK and Src phosphorylation sites on Raf-1 to aspartate, a phosphate mimic, prevented RKIP association with or inhibition of Raf-1 signaling. Interestingly, although RKIP can interact with B-Raf, RKIP depletion had no effect on activation of B-Raf. Because c-Raf-1 and B-Raf are both required for maximal MAPK stimulation by epidermal growth factor in neuronal and epithelial cell lines, we determined whether RKIP significantly affects MAPK signaling. In fact, RKIP depletion increased not only the amplitude but also the sensitivity of MAPK and DNA synthesis to epidermal growth factor stimulation by up to an order of magnitude. These results indicate that selective modulation of c-Raf-1 but not B-Raf activation by RKIP can limit the dynamic range of the MAPK signaling response to growth factors and may play a critical role in growth and development.     

Akt-mediated signaling is induced by cytokines and cyclic adenosine monophosphate and suppresses hepatocyte inducible nitric oxide synthase expression independent of MAPK P44/42

Cyclic AMP inhibits the expression of nitric oxide synthase (Harbrecht et al., 1995 [1]) in hepatocytes but the mechanism for this effect is incompletely understood. Cyclic AMP can activate several intracellular signaling pathways in hepatocytes including Protein Kinase A (PKA), cAMP regulated guanine nucleotide exchange factors (cAMP-GEFs), and calcium-mediated Protein Kinases. There is considerable overlap and cross-talk between many of these signaling pathways, however, and how these cascades regulate hepatocyte iNOS is not known. We hypothesized that Akt mediates the effect of cAMP on hepatocyte iNOS expression. Hepatocytes cultured with cytokines and dbcAMP increased Akt phosphorylation up to 2 h of culture. Akt phosphorylation was inhibited by the PI3K inhibitor LY294002 (10 μM), farnyltranferase inhibitor FTI-276, or transfection with a dominant negative Akt. The cyclic AMP-induced suppression of cytokine-stimulated iNOS was partially reversed by LY294002 and FTI-276. LY294002 also increased NFκB nucleus translocation by Western blot analysis in nuclear extracts. Cyclic AMP increased phosphorylation of Raf1 at serine 259 which was blocked by LY294002 and associated with decreased MAPK P44/42 phosphorylation. However, inhibition of MAPK P44/42 signaling with PD98059 failed to suppress cytokine-induced hepatocyte iNOS expression and did not enhance the inhibitory effect of dbcAMP on iNOS production. A constitutively active MAPK P44/42 plasmid had no effect on cytokine-stimulated NO production. These data demonstrate that dbcAMP regulates hepatocyte iNOS expression through an Akt-mediated signaling mechanism that is independent of MAPK P44/42.     

The inhibitory action of sodium arsenite on lipopolysaccharide-induced nitric oxide production in RAW 267.4 macrophage cells: a role of Raf-1 in lipopolysaccharide signaling

The effect of sodium arsenite (SA) on LPS-induced NO production in RAW 267.4 murine macrophage cells was studied. SA pretreatment of LPS-stimulated RAW cells resulted in a striking reduction in NO production. No significant difference in LPS binding was observed between RAW cells pretreated with SA and control untreated RAW cells, suggesting that SA might impair the intracellular signal pathway for NO production. SA inhibited LPS-induced NF-kappaB activation by preventing loss of IkappaB-alpha and -beta. Furthermore, SA blocked phosphorylation of extracellular signal-regulated kinase 1/2 (Erk1/2), but not phosphorylation of p38 and c-Jun N-terminal kinase. SA treatment resulted in the disappearance of Raf-1, suggesting that it might cause the inhibition of the Erk1/2 mitogen-activated protein (MAP) kinase pathway. The SA-mediated loss of Raf-1 also abolished LPS-induced NF-kappaB activation as well as the Erk1/2 pathway. The dominant negative mutant of MAP kinase kinase 1 inhibited both NO production and NF-kappaB activation in LPS-stimulated RAW cells. Taken together, these results indicate that the inhibitory action of SA on NO production in LPS-stimulated macrophages might be due to abrogation of inducible NO synthase induction, and it might be closely related to inactivation of the NF-kappaB and Erk1/2 MAP kinase pathways through loss of Raf-1.     

Regulation of cyclic adenosine 3',5'-monophosphate-dependent protein kinase activity and regulatory subunit RII beta content by basic fibroblast growth factor (bFGF) during granulosa cell differentiation: possible implication of protein kinase C in bFGF action.

We have previously shown that basic fibroblast growth factor (bFGF) inhibits the FSH-induced differentiation of cultured rat granulosa cells, as manifested by prominent reduction of the LH receptor expression. We now investigate the possible sites and mechanism of action of bFGF. Whereas bFGF decreased the cAMP formation induced by FSH, it enhanced the cAMP production caused by cholera toxin and forskolin, suggesting that bFGF exerted its inhibitory action on cell differentiation at a step to cAMP production. Photoaffinity labeling with 8-azido-[32P]cAMP revealed that bFGF markedly reduced the FSH-induced increase in the level of regulatory subunit RII beta of the cAMP-dependent protein kinase (PKA) type II. In contrast to its striking effect on RII beta expression (70-80% inhibition), bFGF decreased PKA enzymatic activity by only 30%. On the other hand, transforming growth factor-beta (TGF beta) slightly amplified the stimulatory action of FSH and antagonized the bFGF inhibitory effect on both LH receptor expression and RII beta synthesis. We report that the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol-13-acetate (TPA), which impaired granulosa cell differentiation, also abolished the RII beta synthesis induced by FSH. The activation of PKC by bFGF in granulosa cells was supported by the following findings: (i) bFGF markedly enhanced the production of diacylglycerol (2.3-fold stimulation at 5 min), the intracellular activator of PKC; (ii) bFGF promoted tight association of PKC to cellular membranes, a process that is believed to correlate with the enzyme activation; (iii) bFGF induced the phosphorylation of an endogenous M(r) 78,000/pI 4.7 protein that appears as a specific PKC substrate; (iv) bFGF mimicked the TPA-induced transmodulation of the epidermal growth factor (EGF) receptor, reducing by 36% the 125I-EGF binding on granulosa cells. We conclude that bFGF may exert its repressive action on RII beta synthesis, PKA activity, and granulosa cell differentiation by primarily targeting PKC activation.     

Coupling of the insulin-like growth factor-I receptor tyrosine kinase to Gi2 in human intestinal smooth muscle: Gbetagamma -dependent mitogen-activated protein kinase activation and growth

Endogenous insulin-like growth factor-1 (IGF-I) stimulates growth of cultured human intestinal smooth muscle by activating distinct mitogen-activated protein (MAP) kinase-dependent and phosphatidylinositol 3-kinase-dependent signaling pathways. In Rat1 and Balb/c3T3 fibroblasts and in neurons the IGF-I receptor is coupled to an inhibitory G protein, G(i), which mediates G(beta)gamma-dependent MAP kinase activation. The present study determined whether in normal human intestinal smooth muscle cells the IGF-I receptor activates a heterotrimeric G protein and the role of G protein activation in mediating IGF-I-induced growth. IGF-I elicited IGF-I receptor tyrosine phosphorylation, resulting in the specific activation of G(i2). G(beta)gamma subunits selectively mediated IGF-I-dependent MAP kinase activation; G(alpha)i2 subunits selectively mediated IGF-I-dependent inhibition of adenylyl cyclase activity. IGF-I-stimulated MAP kinase activation and growth were inhibited by pertussis toxin, an inhibitor of G(i)/G(o) activation. Cyclic AMP inhibits growth of human intestinal muscle cells. IGF-I inhibited both basal and forskolin-stimulated cAMP levels. This inhibition was attenuated in the presence of pertussis toxin. IGF-I stimulated phosphatidylinositol 3-kinase activation, in contrast to MAP kinase activation, occurred independently of G(i2) activation. These data suggest that IGF-I specifically activates G(i2), resulting in concurrent G(beta)gamma-dependent stimulation of MAP kinase activity and growth, and G(alpha)i2-dependent inhibition of cAMP levels resulting in disinhibition of cAMP-mediated growth suppression.     

Raf-1 protein kinase is important for progesterone-induced Xenopus oocyte maturation and acts downstream of mos.

In somatic cells, the Raf-1 serine/threonine protein kinase is activated by several polypeptide growth factors. We investigated the role of Raf-1 in progesterone-induced meiotic maturation of Xenopus laevis oocytes. Raf-1 enzymatic activity and phosphorylation (reflected by a mobility shift on sodium dodecyl sulfate gels) were increased in oocytes following progesterone stimulation. The increase in Raf-1 activity was concurrent with an elevation in the activity of mitogen-activated protein (MAP) kinase. When RNA encoding an oncogenic form of Raf-1 (v-Raf) was injected into immature oocytes, MAP kinase mobility shift, germinal vesicle breakdown, and histone H1 phosphorylation increased markedly. When RNA encoding a dominant-negative version of Raf-1 was injected, progesterone-induced oocyte maturation was blocked. When RNA encoding Xenopus mos (mosxe) was injected into oocytes, Raf-1 and MAP kinase mobility shifts were observed after several hours. Also, when antisense mosxe oligonucleotides were injected into oocytes, progesterone-induced Raf-1 and MAP kinase mobility shifts were blocked. Finally, when antisense mosxe oligonucleotides were coinjected with v-Raf RNA into oocytes, histone H1 kinase activation, germinal vesicle breakdown, and MAP kinase mobility shift occurred. These findings suggest that Raf-1 activity is required for progesterone-induced oocyte maturation and that Raf-1 is downstream of mosxe activity.     

Molecular aspects of mechanical stress-induced cardiac hypertrophy

To elucidate the signal transduction pathway from external stimuli to nuclear gene expression in mechanical stress-induced cardiac hypertrophy, we examined the time course of activation of protein kinases such as Raf-1 kinase (Raf-1), mitogen-activated protein kinase kinase (MAPKK), MAP kinases (MAPKs) and 90-kDa ribosomal S6 kinase (p90^rsk) in neonatal rat cardiomyocytes. Mechanical stretch rapidly activated Raf-1 and its maximal activation was observed at 1–2 min after stretch. The activity of MAPKK was also increased by stretch, with a peak at 5 min after stretch. In addition, MAPKs and p90^rsk were maximally activated at 8 min and at 10–30 min after stretch, respectively. Next, the relationship between mechanical stress-induced hypertrophy and the cardiac renin-angiotensin system was investigated. When the stretch-conditioned culture medium was transferred to the culture dish of non-stretched cardiac myocytes, the medium activated MAPK activity slightly but significantly, and the activation was completely blocked by the type 1 angiotensin II receptor antagonist, CV-11974. However, activation of Raf-1 and MAPKs provoked by stretching cardiomyocytes was only partially suppressed by pretreatment with CV-11974. These results suggest that mechanical stress activates the protein kinase cascade of phosphorylation in cardiac myocytes in the order of Raf-1, MAPKK, MAPKs and p90^rsk, and that angiotensin II, which is secreted from stretched myocytes, activates a part of these protein kinases.Abbreviations MAPK mitogen-activated protein kinase - MAPKK MAP kinase kinase - Raf-1 - Raf- 1 kinase p90^rsk, 90 kDa ribosomal S6 kinase; AngII - angiotensin II - MAPKKK MAP kinase kinase kinase - rMAPK recombinant MAPKK fused to gluthathione S transferase - MMAKK recombinant MAPK fused to maltose binding protein - MBP myelin basic protein - ACE angiotensin-converting enzyme     

Protein Phosphorylation in Astrocytes Mediated by Protein Kinase C: Comparison with Phosphorylation by Cyclic AMP-Dependent Protein Kinase

The protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), has been found recently to transform cultured astrocytes from flat, polygonal cells into stellate-shaped, process-bearing cells. Studies were conducted to determine the effect of PMA on protein phosphorylation in astrocytes and to compare this pattern of phosphorylation with that elicited by dibutyryl cyclic AMP (dbcAMP), an activator of the cyclic AMP-dependent protein kinase which also affects astrocyte morphology. Exposure to PMA increased the amount of 32P incorporation into several phosphoproteins, including two cytosolic proteins with molecular weights of 30,000 (pI 5.5 and 5.7), an acidic 80,000 molecular weight protein (pI 4.5) present in both the cytosolic and membrane fractions, and two cytoskeletal proteins with molecular weights of 60,000 (pI 5.3) and 55,000 (pI 5.6), identified as vimentin and glial fibrillary acidic protein, respectively. Effects of PMA on protein phosphorylation were not observed in cells depleted of protein kinase C. In contrast to the effect observed with PMA, treatment with dbcAMP decreased the amount of 32P incorporation into the 80,000 protein. Like PMA, treatment with dbcAMP increased the 32P incorporation into the proteins with molecular weights of 60,000, 55,000 and 30,000, although the magnitude of this effect was different. The effect of dbcAMP on protein phosphorylation was still observed in cells depleted of protein kinase C. The results suggest that PMA, via the activation of protein kinase C, can alter the phosphorylation of a number of proteins in astrocytes, and some of these same phosphoproteins are also phosphorylated by the cyclic AMP-dependent mechanisms.     

16K human prolactin inhibits vascular endothelial growth factor-induced activation of Ras in capillary endothelial cells.

Signaling pathways mediating the antiangiogenic action of 16K human (h)PRL include inhibition of vascular endothelial growth factor (VEGF)-induced activation of the mitogen-activated protein kinases (MAPK). To determine at which step 16K hPRL acts to inhibit VEGF-induced MAPK activation, we assessed more proximal events in the signaling cascade. 16K hPRL treatment blocked VEGF-induced Raf-1 activation as well as its translocation to the plasma membrane. 16K hPRL indirectly increased cAMP levels; however, the blockade of Raf-1 activation was not dependent on the stimulation of cAMP-dependent protein kinase (PKA), but rather on the inhibition of the GTP-bound Ras. The VEGF-induced tyrosine phosphorylation of the VEGF receptor, Flk-1, and its association with the Shc/Grb2/Ras-GAP (guanosine triphosphatase-activating protein) complex were unaffected by 16K hPRL treatment. In contrast, 16K hPRL prevented the VEGF-induced phosphorylation and dissociation of Sos from Grb2 at 5 min, consistent with inhibition by 16K hPRL of the MEK/MAPK feedback on Sos. The inhibition of Ras activation was paralleled by the increased phosphorylation of 120 kDa proteins comigrating with Ras-GAP. Taken together, these findings show that 16K hPRL inhibits the VEGF-induced Ras activation; this antagonism represents a novel and potentially important mechanism for the control of angiogenesis.     

Fibroblast Growth Factors Stimulate Protein Tyrosine Phosphorylation and Mitogen-Activated Protein Kinase Activity in Primary Cultures of Hippocampal Neurons

Abstract: Fibroblast growth factors (FGFs) are not only mitogens, but they also promote the differentiation of various cell types. For instance, basic FGF (bFGF) provides a critical trophic support for hippocampal neurons in culture. To elicit their biological effects, FGFs interact with high-affinity receptors that are transmembrane proteins with a cytoplasmic portion containing a tyrosine kinase activity. The tyrosine phosphorylation pattern was examined in primary cultures of hippocampal neurons derived from rat embryos. In these cultures grown for 3 days in the absence of serum, the addition of bFGF causes a rapid increase of tyrosine phosphorylation for various proteins with an optimal level after 5 min of bFGF exposure. Concomitantly, bFGF activates mitogen-activated protein kinase (MAP kinase) activity measured with a selective MAP kinase peptide. The activity increased rapidly after the addition of bFGF and remained elevated even when cultures were treated for 1 h with bFGF. Both acidic and basic FGF were able to enhance protein tyrosine phosphorylation and MAP kinase activity, whereas nerve growth factor and epidermal growth factor did not elicit any of these responses. These data indicate that some of the transduction signals (i.e., tyrosine phosphorylation and activation of MAP kinase) that have been described for the proliferative effect of FGFs are also involved when FGFs act as trophic factors for postmitotic neurons in culture.