Evidence implicating a mid-region sequence of IGFBP-3 in its specific IGF-independent actions

In the present study, we investigated whether the mitogen-activated protein (MAP) kinase superfamily is involved in the bone morphogenetic protein (BMP)-4-stimulated synthesis of osteocalcin in osteoblast-like MC3T3-E1 cells. BMP-4 dose-dependently stimulated osteocalcin synthesis. BMP-4 markedly induced the phosphorylation of p44/p42 MAP kinase and p38 MAP kinase, while having little effect on SAPK (stress-activated protein kinase)/JNK (c-Jun N terminal kinase) phosphorylation. SB203580 and PD169316, specific inhibitors of p38 MAP kinase, significantly reduced the osteocalcin synthesis stimulated by BMP-4. In contrast, PD98059 and U0126, inhibitors of upstream kinase of p44/p42 MAP kinase, markedly enhanced the BMP-4-stimulated osteocalcin synthesis. The BMP-4-induced phosphorylation of p44/p42 MAP kinase was suppressed by PD98059, which did not, however, affect the BMP-4-induced phosphorylation of p38 MAP kinase. Taken together, our results strongly suggest that p38 MAP kinase takes part in BMP-4-stimulated osteocalcin synthesis as a positive regulator in osteoblasts, whereas p44/p42 MAP kinase acts as a negative regulator in the synthesis.     

P38 MAP kinase signaling is required for the conversion of CD4+CD25- T cells into iTreg

CD4+CD25+ regulatory T cells (Treg) are important mediators of immune tolerance. A subset of Treg can be generated in the periphery by TGF-beta dependent conversion of conventional CD4+CD25- T cells into induced Treg (iTreg). In chronic viral infection or malignancy, such induced iTreg, which limit the depletion of aberrant or infected cells, may be of pathogenic relevance. To identify potential targets for therapeutic intervention, we investigated the TGF-beta signaling in Treg. In contrast to conventional CD4+ T cells, Treg exhibited marked activation of the p38 MAP kinase pathway. Inhibition of p38 MAP kinase activity prevented the TGF-beta-dependent conversion of CD4+CD25- T cells into Foxp3+ iTreg in vitro. Of note, the suppressive capacity of nTreg was not affected by inhibiting p38 MAP kinase. Our findings indicate that signaling via p38 MAP kinase seems to be important for the peripheral generation of iTreg; p38 MAP kinase could thus be a therapeutic target to enhance immunity to chronic viral infection or cancer.     

A single amino acid substitution makes ERK2 susceptible to pyridinyl imidazole inhibitors of p38 MAP kinase.

Mitogen-activated protein (MAP) kinases are serine/threonine kinases that mediate intracellular signal transduction pathways. Pyridinyl imidazole compounds block pro-inflammatory cytokine production and are specific p38 kinase inhibitors. ERK2 is related to p38 in sequence and structure, but is not inhibited by pyridinyl imidazole inhibitors. Crystal structures of two pyridinyl imidazoles complexed with p38 revealed these compounds bind in the ATP site. Mutagenesis data suggested a single residue difference at threonine 106 between p38 and other MAP kinases is sufficient to confer selectivity of pyridinyl imidazoles. We have changed the equivalent residue in human ERK2, Q105, into threonine and alanine, and substituted four additional ATP binding site residues. The single residue change Q105A in ERK2 enhances the binding of SB202190 at least 25,000-fold compared to wild-type ERK2. We report enzymatic analyses of wild-type ERK2 and the mutant proteins, and the crystal structure of a pyridinyl imidazole, SB203580, bound to an ERK2 pentamutant, I103L, Q105T, D106H, E109G. T110A. These ATP binding site substitutions induce low nanomolar sensitivity to pyridinyl imidazoles. Furthermore, we identified 5-iodotubercidin as a potent ERK2 inhibitor, which may help reveal the role of ERK2 in cell proliferation.     

Activation of p38 mitogen-activated protein kinase in vivo selectively induces apoptosis of CD8(+) but not CD4(+) T cells

CD4(+) and CD8(+) T cells play specific roles during an immune response. Different molecular mechanisms could regulate the proliferation, death, and effector functions of these two subsets of T cells. The p38 mitogen-activated protein (MAP) kinase pathway is induced by cytokines and environmental stress and has been associated with cell death and cytokine expression. Here we report that activation of the p38 MAP kinase pathway in vivo causes a selective loss of CD8(+) T cells due to the induction of apoptosis. In contrast, activation of p38 MAP kinase does not induce CD4(+) T-cell death. The apoptosis of CD8(+) T cells is associated with decreased expression of the antiapoptotic protein Bcl-2. Regulation of the p38 MAP kinase pathway in T cells is therefore essential for the maintenance of CD4/CD8 homeostasis in the peripheral immune system. Unlike cell death, gamma interferon production is regulated by the p38 MAP kinase pathway in both CD4(+) and CD8(+) T cells. Thus, specific aspects of CD4(+) and CD8(+) T-cell function are differentially controlled by the p38 MAP kinase signaling pathway.     

Stimulation of glucose transport in response to activation of distinct AMPK signaling pathways

AMP-activated protein kinase (AMPK) plays a critical role in the stimulation of glucose transport in response to hypoxia and inhibition of oxidative phosphorylation. In the present study, we examined the signaling pathway(s) mediating the glucose transport response following activation of AMPK. Using mouse fibroblasts of AMPK wild type and AMPK knockout, we documented that the expression of AMPK is essential for the glucose transport response to both azide and 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR). In Clone 9 cells, the stimulation of glucose transport by a combination of azide and AICAR was not additive, whereas there was an additive increase in the abundance of phosphorylated AMPK (p-AMPK). In Clone 9 cells, AMPK wild-type fibroblasts, and H9c2 heart cells, azide or hypoxia selectively increased p-ERK1/2, whereas, in contrast, AICAR selectively stimulated p-p38; phosphorylation of JNK was unaffected. Azide's effect on p-ERK1/2 abundance and glucose transport in Clone 9 cells was partially abolished by the MEK1/2 inhibitor U0126. SB 203580, an inhibitor of p38, prevented the phosphorylation of p38 and the glucose transport response to AICAR and, unexpectedly, to azide. Hypoxia, azide, and AICAR all led to increased phosphorylation of Akt substrate of 160 kDa (AS160) in Clone 9 cells. Employing small interference RNA directed against AS160 did not inhibit the glucose transport response to azide or AICAR, whereas the content of P-AS160 was reduced by approximately 80%. Finally, we found no evidence for coimmunoprecipitation of Glut1 and p-AS160. We conclude that although azide, hypoxia, and AICAR all activate AMPK, the downstream signaling pathways are distinct, with azide and hypoxia stimulating ERK1/2 and AICAR stimulating the p38 pathway.     

Mutagenesis of p38alpha MAP kinase establishes key roles of Phe169 in function and structural dynamics and reveals a novel DFG-OUT state

In order to study the role of Phe169 in p38alpha MAP kinase structure and function, wild-type p38alpha and five p38alpha DFG motif mutants were examined in vitro for phosphorylation by MKK6, kinase activity toward ATF2 substrate, thermal stability, and X-ray crystal structure. All six p38alpha variants were efficiently phosphorylated by MKK6. However, only one activated p38alpha mutant (F169Y) possessed measurable kinase activity (1% compared to wild-type). The loss of kinase activity among the DFG mutants may result from an inability to correctly position Asp168 in the activated form of p38alpha. Two mutations significantly increased the thermal stability of p38alpha (F169A DeltaTm = 1.3 degrees C and D168G DeltaTm = 3.8 degrees C), and two mutations significantly decreased the stability of p38alpha (F169R DeltaTm = -3.2 degrees C and F169G DeltaTm = -4.7 degrees C). Interestingly, X-ray crystal structures of two thermally destabilized p38alpha-F169R and p38alpha-F169G mutants revealed a DFG-OUT conformation in the absence of an inhibitor molecule. This DFG-OUT conformation, termed alpha-DFG-OUT, is different from the ones previously identified in p38alpha crystal structures with bound inhibitors and postulated from high-temperature molecular dynamics simulations. Taken together, these results indicate that Phe169 is optimized for p38alpha functional activity and structural dynamics, rather than for structural stability. The alpha-DFG-OUT conformation observed for p38alpha-F169R and p38alpha-F169G may represent a naturally occurring intermediate state of p38alpha that provides access for binding of allosteric inhibitors. A model of the local forces driving the DFG IN-OUT transition in p38alpha is proposed.     

Activation of p38 MAP kinase by DNA double-strand breaks in V(D)J recombination induces a G2/M cell cycle checkpoint

Delay of cell cycle progression in response to double-strand DNA breaks (DSBs) is critical to allow time for DNA repair and prevent cellular transformation. Here, we show that the p38 mitogen-activated protein (MAP) kinase signaling pathway is activated in immature thymocytes along with TcRbeta gene V(D)J recombination. Active p38 MAP kinase promotes a G2/M cell cycle checkpoint through the phosphorylation and activation of p53 in these cells in vivo. Inactivation of p38 MAP kinase and p53 is required for DN3 thymocytes to exit the G2/M checkpoint, progress through mitosis and further differentiate. We propose that p38 MAP kinase is activated by V(D)J-mediated DSBs and induces a p53-mediated G2/M checkpoint to allow DNA repair and prevent cellular transformation.     

Gadd45alpha regulates p38-dependent dendritic cell cytokine production and Th1 differentiation

Gadd45alpha inhibits the activation of p38 by the T cell alternative pathway involving phosphorylation of p38 Tyr(323). Given that T cell p38 may play a role in Th1 development, the response to Th-skewing Ags was analyzed in Gadd45alpha(-/-) mice. Despite constitutively increased p38 activity in Gadd45alpha(-/-) T cells, the Th1 immune response to Toxoplasma gondii Ag (STAg), was diminished. In contrast to T cells, dendritic cells (DC) lacked the alternative p38 activation pathway. Gadd45alpha(-/-) DCs responded to STAg with low levels of MAP kinase cascade-dependent p38 activation, IL-12 production, and CD40 expression. Wild-type T cells transferred into Gadd45alpha(-/-) recipients had a diminished Th1 response to STAg, whereas Gadd45alpha(-/-) T cells transferred into wild-type hosts behaved normally. Therefore, Gadd45alpha has tissue-specific and opposing functions on p38 activity, and Gadd45alpha-regulated p38 activation in DCs is a critical event in Th1 polarization in vivo.     

Kinetic mechanism and ATP-binding site reactivity of p38gamma MAP kinase

Activated p38gamma MAP kinase exhibited significant basal ATPase activity in the absence of a kinase substrate, and addition of a phosphoacceptor substrate increased k(cat)/K(m)20-fold. AMP-PCP was competitive with ATP binding and non-competitive with phosphoacceptor substrate binding. The nucleotide binding site affinity label 5'-(p-fluorosulfonylbenzoyl)adenosine (FSBA) bound stoichiometrically at Lys-56 in the ATP site of both unphosphorylated and activated p38gamma. AMP-PCP only protected the activated enzyme from FSBA inactivation, implying that AMP-PCP does not bind unphosphorylated p38gamma. Basal ATPase activities were also observed for activated p38alpha, ERK2 and JNK3 suggesting that the enzymatic mechanism may be similar for all classes of MAP kinases.     

p38 MAP kinase regulates BMP-4-stimulated VEGF synthesis via p70 S6 kinase in osteoblasts

We previously reported that p70 S6 kinase takes part in bone morphogenetic protein-4 (BMP-4)-stimulated vascular endothelial growth factor (VEGF) synthesis in osteoblast-like MC3T3-E1 cells. Recently, we showed that BMP-4-induced osteocalcin synthesis is regulated by p44/p42 MAP kinase and p38 MAP kinase in these cells. In the present study, we investigated whether the MAP kinases are involved in the BMP-4-stimulated synthesis of VEGF in MC3T3-E1 cells. PD-98059 and U-0126, inhibitors of the upstream kinase of p44/p42 MAP kinase, failed to affect BMP-4-stimulated VEGF synthesis. SB-203580 and PD-169316, inhibitors of p38 MAP kinase, significantly reduced VEGF synthesis, whereas SB-202474, a negative control for p38 MAP kinase inhibitor, had little effect on VEGF synthesis. The BMP-4-stimulated phosphorylation of p38 MAP kinase was not affected by rapamycin, an inhibitor of p70 S6 kinase. On the contrary, SB-203580 and PD-169316 reduced the BMP-4-stimulated phosphorylation of p70 S6 kinase. In addition, anisomycin, an activator of p38 MAP kinase, phosphorylates p70 S6 kinase, and the phosphorylation was suppressed by SB-203580. LY-294002, an inhibitor of phosphatidylinositol 3-kinase, failed to suppress the phosphorylation of p38 MAP kinase induced by BMP-4. Not BMP-4 but anisomycin weakly induced the phosphorylation of phosphoinositide-dependent kinase-1. However, anisomycin had little effect on phosphorylation of either Akt or the mammalian target of rapamycin. Taken together, our results suggest that p38 MAP kinase functions in BMP-4-stimulated VEGF synthesis as a positive regulator at a point upstream from p70 S6 kinase in osteoblasts.     

Direct phosphorylation of focal adhesion kinase by c-Src: evidence using a modified nucleotide pocket kinase and ATP analog

A single mutation in the nucleotide binding pocket of select protein kinases allows for use of a bulky, substituted-ATP analog not used by the wild-type kinase [1]. Using this approach with the protein tyrosine kinase c-Src, we have generated a mutant T338G and expressed it in Src/Yes/Fyn null fibroblasts (SYF1) at near endogenous levels. T338G Src exhibits high specificity for a substituted ATP analog N(6)-2-phenyl ethyl ATP (peATP), which is not used by wild-type c-Src in autophosphorylation nor substrate phosphorylation assays. By employing the T338G Src mutant and [gamma-(32)P]peATP analog, we demonstrate that c-Src can directly phosphorylate focal adhesion kinase (Fak) in vitro. We also show that incubation of permeabilized, T338 Src-expressing cells with peATP causes an increase in Fak tyrosine phosphorylation not observed in wild-type Src cells. Taken together, these data provide evidence that Src directly phosphorylates Fak and demonstrates the limitations of using this modified ATP strategy for analysis of direct substrates of protein kinases in permeabilized cells.     

BetaPix-enhanced p38 activation by Cdc42/Rac/PAK/MKK3/6-mediated pathway. Implication in the regulation of membrane ruffling

betaPix (PAK-interacting exchange factor) is a recently identified guanine nucleotide exchange factor for Rho family small G protein Cdc42/Rac. The protein interacts with p21-activated protein kinase (PAK) through its SH3 domain. We examined the effect of betaPix on MAP kinase signaling and cytoskeletal rearrangement in NIH3T3 fibroblast cells. Overexpression of betaPix enhanced the activation of p38 in the absence of other stimuli and also induced translocation of p38 to the nucleus. This betaPix-induced p38 activation was blocked by coexpression of dominant-negative Cdc42/Rac or kinase-inactive PAK, indicating that the effect of betaPix on p38 is exerted through the Cdc42/Rac-PAK pathway and requires PAK kinase activity. The essential role of betaPix in growth factor-stimulated p38 activation was evidenced by the blocking of platelet-derived growth factor-induced p38 activation in the cells expressing betaPix SH3m (W43K) and betaPix DHm (L238R,L239R). In addition, SB203580, a p38 inhibitor, and kinase-inactive p38 (T180A,Y182F) blocked membrane ruffling induced by betaPix, suggesting that p38 might be involved in mediating betaPix-induced membrane ruffling. The results in this study suggest that betaPix might have a role in nuclear signaling, as well as in actin cytoskeleton regulation, and that some part of these cellular functions is possibly mediated by p38 MAP kinase.     

Rho-kinase regulates endothelin-1-stimulated IL-6 synthesis via p38 MAP kinase in osteoblasts

We have previously reported that endothelin-1 (ET-1) stimulates interleukin-6 (IL-6), a potent bone resorptive agent, through p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the involvement of Rho-kinase in the ET-1-stimulated IL-6 synthesis in MC3T3-E1 cells. ET-1 time-dependently induced the phosphorylation of myosin phosphatase targeting subunit (MYPT-1), a Rho-kinase substrate. Y27632, a specific inhibitor of Rho-kinase, significantly suppressed the IL-6 synthesis induced by ET-1 as well as the MYPT-1 phosphorylation. Fasudil, another inhibitor of Rho-kinase, reduced the ET-1-stimulated IL-6 synthesis. Y27632 as well as fasudil attenuated the ET-1-induced phosphorylation of p38 MAP kinase but not p44/p42 MAP kinase. These results strongly suggest that Rho-kinase regulates ET-1-stimulated IL-6 synthesis through p38 MAP kinase activation in osteoblasts.     

p38α MAP Kinase C-Terminal Domain Binding Pocket Characterized by Crystallographic and Computational Analyses

The mitogen-activated protein (MAP) kinase protein family has a critical role in cellular signaling events, with MAP kinase p38α acting in inflammatory processes and being an important drug discovery target. MAP kinase drug design efforts have focused on small-molecule inhibitors of the ATP catalytic site, which exhibit dose-limiting adverse effects. Therefore, characterizing other potential sites that bind substrates, inhibitors, or allosteric effectors is of great interest. Here, we present the crystal structure of human p38α MAP kinase, which has a lead compound bound both in the active site and in the lipid-binding site of the C-terminal cap. This C-terminal cap is formed from an extension to the kinase fold, unique to the MAP kinase and cyclin-dependent kinase families and glycogen synthase kinase 3. Binding of this lead, 4-[3-(4-fluorophenyl)-1H-pyrazol-4-yl]pyridine, to wild-type p38α induces movement of the C-terminal cap region, creating a hydrophobic pocket centered around residue Trp197. Computational analysis of this C-terminal domain pocket indicates notable flexibility for potentially binding different-shaped compounds, including lipids, oxidized arachidonic acid species such as leukotrienes, and small-molecule effectors. Furthermore, our structural results defining the open p38α C-lobe pocket provide a detailed framework for the design of novel small molecules with affinities comparable to active-site binders: to bind and potentially modulate the shape and activity of p38α in predetermined ways. Moreover, these results and analyses of p38α suggest strategies for designing specific binding compounds applicable to other MAP kinases, as well as the cyclin-dependent kinase family and glycogen synthase kinase 3β that also utilize the C-terminal insert in their interactions.