Pär Gerwins

MEKKs, GCKs, MLKs, PAKs, TAKs, and Tpls: upstream regulators of the c-Jun amino-terminal kinases?

Regulation of the mitogen-activated protein kinase (MAPK) family members - which include the extracellular response kinases (ERKs), p38/HOG1, and the c-Jun amino-terminal kinases (JNKs) - plays a central role in mediating the effects of diverse stimuli encompassing cytokines, hormones, growth factors and stresses such as osmotic imbalance, heat shock, inhibition of protein synthesis and irradiation. A rapidly increasing number of kinases that activate the JNK pathways has been described recently, including the MAPK/ERK kinase kinases, p21-activated kinases, germinal center kinase, mixed lineage kinases, tumor progression locus 2, and TGF-beta-activated kinase. Thus, regulation of the JNK pathway provides an interesting example of how many different stimuli can converge into regulating pathways critical for the determination of cell fate.

Function of fibroblast growth factors and vascular endothelial growth factors and their receptors in angiogenesis.

Angiogenesis, formation of new vessels from pre-existing ones, results from stimulation of endothelial cells, which line the vessel wall. These cells will leave their resting state and start to digest the basement membrane, proliferate, migrate and eventually differentiate to form a hollow tube. All these steps can be induced by growth factors and this review will focus on two important types of angiogenic growth factors, vascular endothelial growth factor (VEGF; also denoted vascular permeability factor, VPF) and fibroblast growth factor (FGF). Both types of factors bind to cell surface expressed receptors, which are ligand-stimulatable tyrosine kinases. Binding of the growth factors to their receptors leads to activation of the intrinsic tyrosine kinase and signal transduction to downstream signalling cascades. This results in transcriptional changes and biological responses. The molecular aspects of signalling cascades critical for endothelial cell proliferation and migration are beginning to be delineated. In contrast, signalling cascades leading to endothelial cell differentiation remain to be determined. Angiogenesis is essential for a number of physiological events such as embryonic development, ovulation, and wound healing. It has become increasingly clear that a number of diseases depend on angiogenesis. For future development of therapeutic tools, it is important to understand the molecular mechanisms that regulate angiogenesis.

Cloning of a Novel Mitogen-activated Protein Kinase Kinase Kinase, MEKK4, That Selectively Regulates the c-Jun Amino Terminal Kinase Pathway

Mitogen-activated protein kinases (MAPKs) are components of sequential kinase cascades that are activated in response to a variety of extracellular signals. Members of the MAPK family include the extracellular response kinases (ERKs or p42/44MAPK), the c-Jun amino-terminal kinases (JNKs), and the p38/Hog 1 protein kinases. MAPKs are phosphorylated and activated by MAPK kinases (MKKs or MEKs), which in turn are phosphorylated and activated by MKK/MEK kinases (Raf and MKKK/MEKKs). We have isolated two cDNAs encoding splice variants of a novel MEK kinase, MEKK4. The MEKK4 mRNA is widely expressed in mouse tissues and encodes for a protein of approximately 180 kDa. The MEKK4 carboxyl-terminal catalytic domain is approximately 55% homologous to the catalytic domains of MEKKs 1, 2, and 3. The amino-terminal region of MEKK4 has little sequence homology to the previously cloned MEKK proteins. MEKK4 specifically activates the JNK pathway but not ERKs or p38, distinguishing it from MEKKs 1, 2 and 3, which are capable of activating the ERK pathway. MEKK4 is localized in a perinuclear, vesicular compartment similar to the Golgi. MEKK4 binds to Cdc42 and Rac; kinase-inactive mutants of MEKK4 block Cdc42/Rac stimulation of the JNK pathway. MEKK4 has a putative pleckstrin homology domain and a proline-rich motif, suggesting specific regulatory functions different from those of the previously characterized MEKKs.

MEK kinase 1, a substrate for DEVD-directed caspases, is involved in genotoxin-induced apoptosis.

ABSTRACT MEK kinase 1 (MEKK1) is a 196-kDa protein that, in response to genotoxic agents, was found to undergo phosphorylation-dependent activation. The expression of kinase-inactive MEKK1 inhibited genotoxin-induced apoptosis. Following activation by genotoxins, MEKK1 was cleaved in a caspase-dependent manner into an active 91-kDa kinase fragment. Expression of MEKK1 stimulated DEVD-directed caspase activity and induced apoptosis. MEKK1 is itself a substrate for CPP32 (caspase-3). A mutant MEKK1 that is resistant to caspase cleavage was impaired in its ability to induce apoptosis. These findings demonstrate that MEKK1 contributes to the apoptotic response to genotoxins. The regulation of MEKK1 by genotoxins involves its activation, which may be part of survival pathways, followed by its cleavage, which generates a proapoptotic kinase fragment able to activate caspases. MEKK1 and caspases are predicted to be part of an amplification loop to increase caspase activity during apoptosis.

Molecular Cloning of Mitogen-activated Protein/ERK Kinase Kinases (MEKK) 2 and 3 REGULATION OF SEQUENTIAL PHOSPHORYLATION PATHWAYS INVOLVING MITOGEN-ACTIVATED PROTEIN KINASE AND c-Jun KINASE

Mitogen-activated protein/ERK kinase kinases (MEKKs) phosphorylate and activate protein kinases which in turn phosphorylate and activate the p42/44 mitogen-activated protein kinase (MAPK), c-Jun/stress-activated protein kinases (JNKs), and p38/Hog1 kinase. We have isolated the cDNAs for two novel mammalian MEKKs (MEKK 2 and 3). MEKK 2 and 3 encode proteins of 69.7 and 71 kDa, respectively. The kinase domains encoded in the COOH-terminal moiety are 94% conserved; the NH-terminal moieties are approximately 65% homologous, suggesting this region may encode sequences conferring differential regulation of the two kinases. Expression of MEKK 2 or 3 in HEK293 cells results in activation of p42/44 and JNK but not of p38/Hog1 kinase. Immunoprecipitated MEKK 2 phosphorylated the MAP kinase kinases, MEK 1, and JNK kinase. Titration of MEKK 2 and 3 expression in transfection assays indicated that MEKK 2 preferentially activated JNK while MEKK 3 preferentially activated p42/44. These findings define a family of MEKK proteins capable of regulating sequential protein kinase pathways involving MAPK members.

p38 MAP kinase negatively regulates endothelial cell survival, proliferation, and differentiation in FGF-2-stimulated angiogenesis

The p38 mitogen–activated protein kinase (p38) is activated in response to environmental stress and inflammatory cytokines. Although several growth factors, including fibroblast growth factor (FGF)-2, mediate activation of p38, the consequences for growth factor–dependent cellular functions have not been well defined. We investigated the role of p38 activation in FGF-2–induced angiogenesis. In collagen gel cultures, bovine capillary endothelial cells formed tubular growth-arrested structures in response to FGF-2. In these collagen gel cultures, p38 activation was induced more potently by FGF-2 treatment compared with that in proliferating cultures. Treatment with the p38 inhibitor SB202190 enhanced FGF-2–induced tubular morphogenesis by decreasing apoptosis, increasing DNA synthesis and cell proliferation, and enhancing the kinetics of cell differentiation including increased expression of the Notch ligand Jagged1. Overexpression of dominant negative mutants of the p38-activating kinases MKK3 and MKK6 also supported FGF-2–induced tubular morphogenesis. Sustained activation of p38 by FGF-2 was identified in vascular endothelial cells in vivo in the chick chorioallantoic membrane (CAM). SB202190 treatment enhanced FGF-2–induced neovascularization in the CAM, but the vessels displayed abnormal features indicative of hyperplasia of endothelial cells. These results implicate p38 in organization of new vessels and suggest that p38 is an essential regulator of FGF-2–driven angiogenesis.

Biomechanical regulation of blood vessel growth during tissue vascularization

Formation of new vessels in granulation tissue during wound healing has been assumed to occur solely through sprouting angiogenesis. In contrast, we show here that neovascularization can be accomplished by nonangiogenic expansion of preexisting vessels. Using neovascularization models based on the chick chorioallantoic membrane and the healing mouse cornea, we found that tissue tension generated by activated fibroblasts or myofibroblasts during wound contraction mediated and directed translocation of the vasculature. These mechanical forces pulled vessels from the preexisting vascular bed as vascular loops with functional circulation that expanded as an integral part of the growing granulation tissue through vessel enlargement and elongation. Blockade of vascular endothelial growth factor receptor-2 confirmed that biomechanical forces were sufficient to mediate the initial vascular growth independently of endothelial sprouting or proliferation. The neovascular network was further remodeled by splitting, sprouting and regression of individual vessels. This model explains the rapid appearance of large functional vessels in granulation tissue during wound healing.

Activation of mitogen-activated protein kinase cascades during priming of human neutrophils by TNF-alpha and GM-CSF.

The signal transduction pathways activated by tumor necrosis factor α (TNF‐α) and granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) that lead to priming of polymorphonuclear leukocytes (PMNs) are unknown. The hypotheses that these cytokines stimulate multiple mitogen‐activated protein kinase (MAPK) cascades, including extracellular signal‐regulated kinases (ERKs), c‐Jun amino‐terminal kinases (JNKs), and p38 MAPK, and that these MAPKs participate in priming of human PMNs were examined. TNF‐α stimulated a dose‐dependent increase in ERK and p38 MAPK activities that was maximal at 10 min. JNKs were not stimulated by TNF‐α or GM‐CSF. GM‐CSF stimulated ERK activity comparable to that of TNF‐α, but GM‐CSF was a less potent stimulus of p38 MAPK activity. The tyrosine kinase inhibitor, genistein, inhibited ERK and p38 MAPK stimulation by both cytokines. The phosphatidylinositol 3‐kinase inhibitor, wortmannin, attenuated stimulation of ERKs and p38 MAPK by GM‐CSF, but not TNF‐α. GM‐CSF, but not TNF‐α, stimulated wortmannin‐sensitive activation of Raf‐1. TNF‐α and GM‐CSF priming of superoxide release stimulated by N‐formyl‐methionyl‐leucyl‐phenylalanine was significantly attenuated by the MEK inhibitor, PD098059, and the p38 MAPK inhibitor, SB203580. Incubation with both MAPK inhibitors produced an additive effect. Our data suggest that TNF‐α and GM‐CSF activate ERKs and p38 MAPK by different signal transduction pathways. Both ERK and p38 MAPK cascades contribute to the ability of TNF‐α and GM‐CSF to prime the respiratory burst response in human PMNs. J. Leukoc. Biol. 64: 537–545; 1998.

Inactivation of Src family kinases inhibits angiogenesis in vivo: implications for a mechanism involving organization of the actin cytoskeleton

Inhibition of angiogenesis could be a treatment strategy for diseases such as cancer, rheumatoid arthritis, and diabetic retinopathy. PP2 is a pharmacological inhibitor of Src family kinases and was found to inhibit FGF-2 induced angiogenesis in vivo. Experiments in vitro showed that PP2 inhibited invasive growth and sprouting of both endothelial and vascular smooth muscle cells into a fibrin matrix. PP2 inhibited the formation of lamellopodia and expression of kinase inactive c-Src reduced phosphorylation of cortactin and paxillin, suggesting a model in which Src kinases are involved in organization of the actin cytoskeleton. Consequently, endothelial cells expressing kinase inactive c-Src failed to spread and form cord-like structures on a collagen matrix. These data suggest that pharmacological inactivation of Src family kinases inhibits FGF-2 stimulated angiogenesis by interference with organization of the actin cytoskeleton in both endothelial and vascular smooth muscle cells, which affects cell migration.

Activation of mitogen-activated protein kinases in experimental cerebral ischemia

Lennmyr F, Karlsson S, Gerwins P, Ata KA, Terént A. Activation of mitogen‐activated protein kinases in experimental cerebral ischemia. Acta Neurol Scand 2002: 106: 333–340.