Role of the JIP4 scaffold protein in the regulation of mitogen-activated protein kinase signaling pathways

The c-Jun NH2-terminal kinase (JNK)-interacting protein (JIP) group of scaffold proteins (JIP1, JIP2, and JIP3) can interact with components of the JNK signaling pathway and potently activate JNK. Here we describe the identification of a fourth member of the JIP family. The primary sequence of JIP4 is most closely related to that of JIP3. Like other members of the JIP family of scaffold proteins, JIP4 binds JNK and also the light chain of the microtubule motor protein kinesin-1. However, the function of JIP4 appears to be markedly different from other JIP proteins. Specifically, JIP4 does not activate JNK signaling. In contrast, JIP4 serves as an activator of the p38 mitogen-activated protein (MAP) kinase pathway by a mechanism that requires the MAP kinase kinases MKK3 and MKK6. The JIP4 scaffold protein therefore appears to be a new component of the p38 MAP kinase signaling pathway.     

Interaction of Rac exchange factors Tiam1 and Ras-GRF1 with a scaffold for the p38 mitogen-activated protein kinase cascade

Tiam1 and Ras-GRF1 are guanine nucleotide exchange factors (GEFs) that activate the Rac GTPase. The two GEFs have similar N-terminal regions containing pleckstrin homology domains followed by coiled-coils and additional sequences that function together to allow regulated GEF activity. Here we show that this N-terminal region of both proteins binds to the scaffold protein IB2/JIP2. IB2/JIP2 is a scaffold for the p38 mitogen-activated protein (MAP) kinase cascade because it binds to the Rac target MLK3, the MAP kinase kinase MKK3, and the p38 MAP kinase. Expression of IB2/JIP2 in cells potentiates the ability of Tiam1 or Ras-GRF1 to activate the p38 MAP kinase cascade but not the Jnk MAP kinase cascade. In addition, Tiam1 or Ras-GRF1 binding to IB2/JIP2 increases the association of the components of the p38 MAP kinase signaling cassette with IB2/JIP2 in cells and activates scaffold-associated p38. These findings imply that Tiam1 and Ras-GRF1 can contribute to Rac signaling specificity by their ability to form a complex with a scaffold that binds components of one of the many known Rac effector pathways.     

Phosphorylation-dependent scaffolding role of JSAP1/JIP3 in the ASK1-JNK signaling pathway. A new mode of regulation of the MAP kinase cascade

JSAP1 (also termed JIP3) is a scaffold protein that interacts with specific components of the JNK signaling pathway. Apoptosis signal-regulating kinase (ASK) 1 is a MAP kinase kinase kinase that activates the JNK and p38 mitogen-activated protein (MAP) kinase cascades in response to environmental stresses such as reactive oxygen species. Here we show that JSAP1 bound ASK1 and enhanced ASK1- and H(2)O(2)-induced JNK activity. ASK1 phosphorylated JSAP1 in vitro and in vivo, and the phosphorylation facilitated interactions of JSAP1 with SEK1/MKK4, MKK7 and JNK3. Furthermore, ASK1-dependent phosphorylation was required for JSAP1 to recruit and thereby activate JNK in response to H(2)O(2). We thus conclude that JSAP1 functions not only as a simple scaffold, but it dynamically participates in signal transduction by forming a phosphorylation-dependent signaling complex in the ASK1-JNK signaling module.     

Activation of p21-activated kinase 6 by MAP kinase kinase 6 and p38 MAP kinase

The p21-activated kinases (PAKs) contain an N-terminal Cdc42/Rac interactive binding domain, which in the group 1 PAKs (PAK1, 2, and 3) regulates the activity of an adjacent conserved autoinhibitory domain. In contrast, the group 2 PAKs (PAK4, 5, and 6) lack this autoinhibitory domain and are not activated by Cdc42/Rac binding, and the mechanisms that regulate their kinase activity have been unclear. This study found that basal PAK6 kinase activity was repressed by a p38 mitogen-activated protein (MAP) kinase antagonist and could be strongly stimulated by constitutively active MAP kinase kinase 6 (MKK6), an upstream activator of p38 MAP kinases. Mutation of a consensus p38 MAP kinase target site at serine 165 decreased PAK6 kinase activity. Moreover, PAK6 was directly activated by MKK6, and mutation of tyrosine 566 in a consensus MKK6 site (threonine-proline-tyrosine, TPY) in the activation loop of the PAK6 kinase domain prevented activation by MKK6. PAK6 activation by MKK6 was also blocked by mutation of an autophosphorylated serine (serine 560) in the PAK6 activation loop, indicating that phosphorylation of this site is necessary for MKK6-mediated activation. PAK4 and PAK5 were similarly activated by MKK6, consistent with a conserved TPY motif in their activation domains. The activation of PAK6 by both p38 MAP kinase and MKK6 suggests that PAK6 plays a role in the cellular response to stress-related signals.     

The mAKAP signaling complex: integration of cAMP, calcium, and MAP kinase signaling pathways

Following its production by adenylyl cyclases, the second messenger cAMP is in involved in pleiotrophic signal transduction. The effectors of cAMP include the cAMP-dependent protein kinase (PKA), the guanine nucleotide exchange factor Epac (exchange protein activated by cAMP), and cAMP-dependent ion channels. In turn, cAMP signaling is attenuated by phosphodiesterase-catalyzed degradation. The association of cAMP effectors and the enzymes that regulate cAMP concentration into signaling complexes helps to explain the differential signaling initiated by members of the G(s)-protein coupled receptor family. The signal transduction complex formed by the scaffold protein mAKAP (muscle A kinase-anchoring protein) at the nuclear envelope of both striated myocytes and neurons contains three cAMP-binding proteins, PKA, Epac1, and the phosphodiesterase PDE4D3. In addition, the mAKAP complex also contains components of the ERK5 MAP kinase signaling pathway, the calcium release channel ryanodine receptor and the phosphatases PP2A as well as calcineurin. Analysis of the mAKAP complex illustrates how a macromolecular complex can serve as a node in the intracellular signaling network of cardiac myocytes to integrate multiple cAMP signals with those of calcium and MAP kinases to regulate the hypertrophic actions of several hormones.     

The JIP Group of Mitogen-Activated Protein Kinase Scaffold Proteins

Activation of the c-Jun NH(2)-terminal kinase (JNK) group of mitogen-activated protein (MAP) kinases is mediated by a protein kinase cascade. This signaling mechanism may be coordinated by the interaction of components of the protein kinase cascade with scaffold proteins. The JNK-interacting protein (JIP) group of scaffold proteins selectively mediates signaling by the mixed-lineage kinase (MLK)-->MAP kinase kinase 7 (MKK7)-->JNK pathway. The scaffold proteins JIP1 and JIP2 interact to form oligomeric complexes that accumulate in peripheral cytoplasmic projections extended at the cell surface. The JIP proteins function by aggregating components of a MAP kinase module (including MLK, MKK7, and JNK) and facilitate signal transmission by the protein kinase cascade.     

MKK6/3 and p38 MAPK pathway activation is not necessary for insulin-induced glucose uptake but regulates glucose transporter expression

p38 mitogen-activated protein kinase (MAPK), which is situated downstream of MAPK kinase (MKK) 6 and MKK3, is activated by mitogenic or stress-inducing stimuli, as well as by insulin. To clarify the role of the MKK6/3-p38 MAPK pathway in the regulation of glucose transport, dominant negative p38 MAPK and MKK6 mutants and constitutively active MKK6 and MKK3 mutants were overexpressed in 3T3-L1 adipocytes and L6 myotubes using an adenovirus-mediated transfection procedure. Constitutively active MKK6/3 mutants up-regulated GLUT1 expression and down-regulated GLUT4 expression, thereby significantly increasing basal glucose transport but diminishing transport induced by insulin. Similar effects were elicited by chronic (24 h) exposure to tumor necrosis factor alpha, interleukin-1beta, or 200 mm sorbitol, all activate the MKK6/3-p38 MAPK pathway. SB203580, a specific p38 MAPK inhibitor, attenuated these effects, further confirming that both MMK6 and MMK3 act via p38 MAPK, whereas they had no effect on the increase in glucose transport induced by a constitutively active MAPK kinase 1 (MEK1) mutant or by myristoylated Akt. In addition, suppression of p38 MAPK activation by overexpression of a dominant negative p38 MAPK or MKK6 mutant did not diminish insulin-induced glucose uptake by 3T3-L1 adipocytes. It is thus apparent that activation of p38 MAPK is not essential for insulin-induced increases in glucose uptake. Rather, p38 MAPK activation leads to a marked down-regulation of insulin-induced glucose uptake via GLUT4, which may underlie cellular stress-induced insulin resistance caused by tumor necrosis factor alpha and other factors.     

Negative feedback regulation of MKK6 mRNA stability by p38alpha mitogen-activated protein kinase

p38 mitogen-activated protein (MAP) kinases play an important role in the regulation of cellular responses to all kinds of stresses. The most abundant and broadly expressed p38 MAP kinase is p38alpha, which can also control the proliferation, differentiation, and survival of several cell types. Here we show that the absence of p38alpha correlates with the up-regulation of one of its upstream activators, the MAP kinase kinase MKK6, in p38alpha(-/-) knockout mice and in cultured cells derived from them. In contrast, the expression levels of the p38 activators MKK3 and MKK4 are not affected in p38alpha-deficient cells. The increase in MKK6 protein concentration correlates with increased amounts of MKK6 mRNA in the p38alpha(-/-) cells. Pharmacological inhibition of p38alpha also up-regulates MKK6 mRNA levels in HEK293 cells. Conversely, reintroduction of p38alpha into p38alpha(-/-) cells reduces the levels of MKK6 protein and mRNA to the normal levels found in wild-type cells. Moreover, we show that the MKK6 mRNA is more stable in p38alpha(-/-) cells and that the 3'untranslated region of this mRNA can differentially regulate the stability of the lacZ reporter gene in a p38alpha-dependent manner. Our data indicate that p38alpha can negatively regulate the stability of the MKK6 mRNA and thus control the steady-state concentration of one of its upstream activators.     

Phosphorylation of the kinase interaction motif in mitogen-activated protein (MAP) kinase phosphatase-4 mediates cross-talk between protein kinase A and MAP kinase signaling pathways

MAP kinase phosphatase 4 (DUSP9/MKP-4) plays an essential role during placental development and is one of a subfamily of three closely related cytoplasmic dual-specificity MAPK phosphatases, which includes the ERK-specific enzymes DUSP6/MKP-3 and DUSP7/MKP-X. However, unlike DUSP6/MKP-3, DUSP9/MKP-4 also inactivates the p38α MAP kinase both in vitro and in vivo. Here we demonstrate that inactivation of both ERK1/2 and p38α by DUSP9/MKP-4 is mediated by a conserved arginine-rich kinase interaction motif located within the amino-terminal non-catalytic domain of the protein. Furthermore, DUSP9/MKP-4 is unique among these cytoplasmic MKPs in containing a conserved PKA consensus phosphorylation site (55)RRXSer-58 immediately adjacent to the kinase interaction motif. DUSP9/MKP-4 is phosphorylated on Ser-58 by PKA in vitro, and phosphorylation abrogates the binding of DUSP9/MKP-4 to both ERK2 and p38α MAP kinases. In addition, although mutation of Ser-58 to either alanine or glutamic acid does not affect the intrinsic catalytic activity of DUSP9/MKP-4, phospho-mimetic (Ser-58 to Glu) substitution inhibits both the interaction of DUSP9/MKP-4 with ERK2 and p38α in vivo and its ability to dephosphorylate and inactivate these MAP kinases. Finally, the use of a phospho-specific antibody demonstrates that endogenous DUSP9/MKP-4 is phosphorylated on Ser-58 in response to the PKA agonist forskolin and is also modified in placental tissue. We conclude that DUSP9/MKP-4 is a bona fide target of PKA signaling and that attenuation of DUSP9/MKP-4 function can mediate cross-talk between the PKA pathway and MAPK signaling through both ERK1/2 and p38α in vivo.     

TAB-1 modulates intracellular localization of p38 MAP kinase and downstream signaling

Stress-activated mitogen-activated protein (MAP) kinase p38 mediates stress signaling in mammalian cells via threonine and tyrosine phosphorylation in its conserved TGY motif by upstream MAP kinase kinases (MKKs). In addition, p38 MAP kinase can also be activated by an MKK-independent mechanism involving TAB-1 (TAK-1-binding protein)-mediated autophosphorylation. Although TAB-1-mediated p38 activation has been implicated in ischemic heart, the biological consequences and downstream signaling of TAB-1-mediated p38 activation in cardiomyocytes is largely unknown. We show here that TAB-1 expression leads to a significant induction of p38 autophosphorylation and consequent kinase activation in cultured neonatal cardiomyocytes. In contrast to MKK3-induced p38 kinase downstream effects, TAB-1-induced p38 kinase activation does not induce expression of pro-inflammatory genes, cardiac marker gene expression, or changes in cellular morphology. Rather, TAB-1 binds to p38 and prevents p38 nuclear localization. Furthermore, TAB-1 disrupts p38 interaction with MKK3 and redirects p38 localization in the cytosol. Consequently, TAB-1 expression antagonizes the downstream activity of p38 kinase induced by MKK3 and attenuates interleukin-1beta-induced inflammatory gene induction in cardiomyocytes. These data suggest that TAB-1 can mediate MKK-independent p38 kinase activation while negatively modulating MKK-dependent p38 function. Our study not only redefines the functional role of TAB-1 in p38 kinase-mediated signaling pathways but also provides the first evidence that intracellular localization of p38 kinase and complex interaction dictates its downstream effects. These results suggest a previously unknown mechanism for stress-MAP kinase regulation in mammalian cells.     

A novel regulatory mechanism of MAP kinases activation and nuclear translocation mediated by PKA and the PTP-SL tyrosine phosphatase

Protein tyrosine phosphatase PTP-SL retains mitogen-activated protein (MAP) kinases in the cytoplasm in an inactive form by association through a kinase interaction motif (KIM) and tyrosine dephosphorylation. The related tyrosine phosphatases PTP-SL and STEP were phosphorylated by the cAMP-dependent protein kinase A (PKA). The PKA phosphorylation site on PTP-SL was identified as the Ser(231) residue, located within the KIM. Upon phosphorylation of Ser(231), PTP-SL binding and tyrosine dephosphorylation of the MAP kinases extracellular signal-regulated kinase (ERK)1/2 and p38alpha were impaired. Furthermore, treatment of COS-7 cells with PKA activators, or overexpression of the Calpha catalytic subunit of PKA, inhibited the cytoplasmic retention of ERK2 and p38alpha by wild-type PTP-SL, but not by a PTP-SL S231A mutant. These findings support the existence of a novel mechanism by which PKA may regulate the activation and translocation to the nucleus of MAP kinases.     

Galectin-3 expression in macrophages is signaled by Ras/MAP kinase pathway and up-regulated by modified lipoproteins

To study the signaling pathway involved in the regulation of galectin-3 expression we used phorbol ester to stimulate macrophage differentiation of THP-1 cells. Treatment with phorbol 12-myristate 13-acetate (PMA) increased significantly the level of expression of galectin-3 in THP-1 cells. PMA-induced galectin-3 overexpression was blocked by: protein kinase C inhibitors staurosporine, calphostin C, and apigenin; tyrosine-specific protein kinase inhibitors genistein and tyrphostin A25; PD 98059, a selective inhibitor of mitogen-activated protein kinase (MAPK) kinase 1 (MEK1 or MKK1); and SB 203580, a specific inhibitor of p38 MAPK. Galectin-3 up-regulation was not affected by exposure to two inhibitors of cAMP-dependent protein kinase (PKA), H-89 and KT5720. Co-transfection of pPG3.5, a plasmid vector containing the rabbit galectin-3 promoter and the constructs pMCL-MKK1 N3 or pRC-RSV-MKK3Glu that constitutively express MKK1 and MKK3, raised the activity of galectin-3 promoter by 185% and 110%, respectively. Co-transfection with a Ha-Ras expression vector stimulated galectin-3 promoter activity approximately 10-fold. Expression of c-Jun or v-Jun raised the level of galectin-3 promoter activity more the three- and fourfold, respectively. Co-transfection of c-Jun and pPG3.5 5'-upstream deletion mutants resulted in a reduction of the galectin-3 promoter activity by 50% to 80%. Transfection of c-Jun, v-Jun or Ha-Ras increased significantly galectin-3 protein in THP-1 cells. These findings indicated that Ras/MEKK1/MKK1-dependent/AP-1 signal transduction pathway plays an important role in the expression of galectin-3 in PMA-stimulated macrophages. We further investigated the effect of modified lipoproteins on galectin-3 expression in macrophages. Murine resident peritoneal macrophages loaded with acetylated low-density lipoprotein (AcLDL) or oxidized LDL (OxLDL) showed increased galectin-3 protein and mRNA. These results showed that treatment of macrophages with PMA or modified lipoproteins results in galectin-3 overexpression. These findings may explain the enhanced expression of galectin-3 in atherosclerotic foam cells and suggest that Ras/MAPK signal transduction pathway is involved in controlling this gene.     

Possible involvement of p38 MAP kinase in prostaglandin E1-induced ALP activity in osteoblast-like cells

Prostaglandins are now recognized to be important regulators for both bone formation and resorption. Among them, prostaglandin E(1) (PGE(1)) has been reported to stimulate cAMP accumulation and to induce alkaline phosphatase (ALP) activity, a marker of differentiation, in osteoblast-like cells. Recently, we have shown that p38 mitogen-activated protein (MAP) kinase pathway regulates ALP activity in response to activation of Gi protein-coupled receptors in mouse osteoblast-like MC3T3-E1 cells (Suzuki et al., Endocrinology 140 (1999) 3177). In the present study, we investigated whether p38 MAP kinase is involved in ALP activation by PGE(1) in MC3T3-E1 osteoblast-like cells. PGE(1) dose-dependently enhanced ALP activities in the concentration range between 1 nM and 1 microM in MC3T3-E1 cells. SB203580, a specific inhibitor of p38 MAP kinase, blocked the increase in ALP activity induced by PGE(1). Further analysis with western blotting suggested that PGE(1) induced an increase in tyrosine (Tyr) phosphorylation of p38 MAP kinase. Both Bt(2)cAMP, a permeable analogue of cAMP, and forskolin, which directly activates adenylate cyclase, also induced an increase in Tyr phosphorylation of p38 MAP kinase. H-89, a potent inhibitor of protein kinase A (PKA), significantly suppressed PGE(1)-induced Tyr phosphorylation of p38 MAP kinase. The results of this study suggest that PGE(1) stimulates p38 MAP kinase through the activation of PKA, resulting in the enhancement of ALP activity.     

Adenovirus-activated PKA and p38/MAPK pathways boost microtubule-mediated nuclear targeting of virus

Nuclear targeting of adenovirus is mediated by the microtubule-dependent, minus-end-directed motor complex dynein/dynactin, in competition with plus- end-directed motility. We demonstrate that adenovirus transiently activates two distinct signaling pathways to enhance nuclear targeting. The first pathway activates integrins and cAMP-dependent protein kinase A (PKA). The second pathway activates the p38/MAP kinase and the downstream MAPKAP kinase 2 (MK2), dependent on the p38/MAPK kinase MKK6, but independent of integrins and PKA. Motility measurements in PKA-inhibited, p38-inhibited or MK2-lacking (MK2(-/-)) cells indicate that PKA and p38 stimulated both the frequency and velocity of minus-end-directed viral motility without affecting the perinuclear localization of transferrin-containing endosomal vesicles. p38 also suppressed lateral viral motilities and MK2 boosted the frequency of minus-end-directed virus transport. Nuclear targeting of adenovirus was rescued in MK2(-/-) cells by overexpression of hsp27, an MK2 target that enhances actin metabolism. Our results demonstrate that complementary activities of PKA, p38 and MK2 tip the transport balance of adenovirus towards the nucleus and thus enhance infection.     

Prostaglandin E2 enhances osteoclastic differentiation of precursor cells through protein kinase A-dependent phosphorylation of TAK1

Prostaglandin E2 (PGE2) synergistically enhances the receptor activator for NF-kappa B ligand (RANKL)-induced osteoclastic differentiation of the precursor cells. Here we investigated the mechanisms of the stimulatory effect of PGE2 on osteoclast differentiation. PGE2 enhanced osteoclastic differentiation of RAW264.7 cells in the presence of RANKL through EP2 and EP4 prostanoid receptors. RANKL-induced degradation of I kappa B alpha and phosphorylation of p38 MAPK and c-Jun N-terminal kinase in RAW264.7 cells were up-regulated by PGE2 in a cAMP-dependent protein kinase A (PKA)-dependent manner, suggesting that EP2 and EP4 signals cross-talk with RANK signals. Transforming growth factor beta-activated kinase 1 (TAK1), an important MAPK kinase kinase in several cytokine signals, possesses a PKA recognition site at amino acids 409-412. PKA directly phosphorylated TAK1 in RAW264.7 cells transfected with wild-type TAK1 but not with the Ser412 --> Ala mutant TAK1. Ser412 --> Ala TAK1 served as a dominant-negative mutant in PKA-enhanced degradation of I kappa B alpha, phosphorylation of p38 MAPK, and PGE2-enhanced osteoclastic differentiation in RAW264.7 cells. Furthermore, forskolin enhanced tumor necrosis factor alpha-induced I kappa B alpha degradation, p38 MAPK phosphorylation, and osteoclastic differentiation in RAW264.7 cells. Ser412 --> Ala TAK1 abolished the stimulatory effects of forskolin on those cellular events induced by tumor necrosis factor alpha. Ser412 --> Ala TAK1 also inhibited the forskolin-induced up-regulation of interleukin 6 production in RAW264.7 cells treated with lipopolysaccharide. These results suggest that the phosphorylation of the Ser412 residue in TAK1 by PKA is essential for cAMP/PKA-induced up-regulation of osteoclastic differentiation and cytokine production in the precursor cells.