Loss of mitogen-activated protein kinase phosphatase-1 protects from hepatic steatosis by repression of cell death-inducing DNA fragmentation factor A (DFFA)-like effector C (CIDEC)/fat-specific protein 27

The integration of metabolic signals required for the regulation of hepatic lipid homeostasis is complex. Previously, we showed that mice lacking expression of the mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) have increased fatty acid oxidation and are protected from the development of hepatic steatosis. Here, we show that leptin receptor-deficient (db/db) mice lacking MKP-1 are also resistant to the development of hepatic steatosis. Microarray analyses of livers from db/db mice lacking MKP-1 showed suppression of peroxisome proliferator-activated receptor γ (PPARγ) target genes. We identified the fat-specific protein 27 (Fsp27), which promotes PPARγ-mediated hepatic steatosis, as repressed in livers of both db/db and high fat diet-fed mice lacking MKP-1. Hepatocytes from MKP-1-deficient mice exhibited reduced PPARγ-induced lipid droplet formation. Mechanistically, loss of MKP-1 inhibited PPARγ function by increasing MAPK-dependent phosphorylation on PPARγ at its inhibitory residue of serine 112. These results demonstrate that in addition to inhibiting hepatic fatty acid oxidation, MKP-1 promotes hepatic lipogenic gene expression through PPARγ. Hence, MKP-1 plays an important role in MAPK-mediated control of hepatic lipid homeostasis.     

Mitogen-activated protein kinase (MAPK) phosphatase 3-mediated cross-talk between MAPKs ERK2 and p38alpha

MAPK phosphatase 3 (MKP3) is highly specific for ERK1/2 inactivation via dephosphorylation of both phosphotyrosine and phosphothreonine critical for enzymatic activation. Here, we show that MKP3 is able to effectively dephosphorylate the phosphotyrosine, but not phosphothreonine, in the activation loop of p38α in vitro and in intact cells. The catalytic constant of the MKP3 reaction for p38α is comparable with that for ERK2. Remarkably, MKP3, ERK2, and phosphorylated p38α can form a stable ternary complex in solution, and the phosphatase activity of MKP3 toward p38α substrate is allosterically regulated by ERK2-MKP3 interaction. This suggests that MKP3 not only controls the activities of ERK2 and p38α but also mediates cross-talk between these two MAPK pathways. The crystal structure of bisphosphorylated p38α has been determined at 2.1 Å resolution. Comparisons between the phosphorylated MAPK structures reveal the molecular basis of MKP3 substrate specificity.     

Signal integration and coincidence detection in the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) cascade: concomitant activation of receptor tyrosine kinases and of LRP-1 leads to sustained ERK phosphorylation via down-regulation of dual specificity phosphatases (DUSP1 and -6)

Diverse stimuli can feed into the MAPK/ERK cascade; this includes receptor tyrosine kinases, G protein-coupled receptors, integrins, and scavenger receptors (LDL receptor-related protein (LRP)). Here, we investigated the consequence of concomitant occupancy of the receptor tyrosine kinases (by EGF, basic FGF, VEGF, etc.) and of LRP family members (by LDL or lactoferrin). The simultaneous stimulation of a receptor tyrosine kinase by its cognate ligand and of LRP-1 (by lactoferrin or LDL) resulted in sustained activation of ERK, which was redirected to the cytoplasm. Accordingly, elevated levels of active cytosolic ERK were translated into accelerated adhesion to vitronectin. The sustained ERK response was seen in several cell types, but it was absent in cells deficient in LRP-1 (but not in cells lacking the LDL receptor). This response was also contingent on the presence of urokinase (uPA) and its receptor (uPAR), because it was absent in uPA(-/-) and uPAR(-/-) fibroblasts. Combined stimulation of the EGF receptor and of LRP-1 delayed nuclear accumulation of phosphorylated ERK. This shift in favor of cytosolic accumulation of phospho-ERK was accounted for by enhanced proteasomal degradation of dual specificity phosphatases DUSP1 and DUSP6, which precluded dephosphorylation of cytosolic ERK. These observations demonstrate that the ERK cascade can act as a coincidence detector to decode the simultaneous engagement of a receptor tyrosine kinase and of LRP-1 and as a signal integrator that encodes this information in a spatially and temporally distinct biological signal. In addition, the findings provide an explanation of why chronic elevation of LRP-1 ligands (e.g. PAI-1) can predispose to cancer.     

Structural Basis for the Regulation of the Mitogen-activated Protein (MAP) Kinase p38α by the Dual Specificity Phosphatase 16 MAP Kinase Binding Domain in Solution

Mitogen-activated protein kinases (MAPKs) fulfill essential biological functions and are key pharmaceutical targets. Regulation of MAPKs is achieved via a plethora of regulatory proteins including activating MAPKKs and an abundance of deactivating phosphatases. Although all regulatory proteins use an identical interaction site on MAPKs, the common docking and hydrophobic pocket, they use distinct kinase interaction motif (KIM or D-motif) sequences that are present in linear, peptide-like, or well folded protein domains. It has been recently shown that a KIM-containing MAPK-specific dual specificity phosphatase DUSP10 uses a unique binding mode to interact with p38α. Here we describe the interaction of the MAPK binding domain of DUSP16 with p38α and show that despite belonging to the same dual specificity phosphatase (DUSP) family, its interaction mode differs from that of DUSP10. Indeed, the DUSP16 MAPK binding domain uses an additional helix, α-helix 4, to further engage p38α. This leads to an additional interaction surface on p38α. Together, these structural and energetic differences in p38α engagement highlight the fine-tuning necessary to achieve MAPK specificity and regulation among multiple regulatory proteins.     

Distinct signaling properties of mitogen-activated protein kinase kinases 4 (MKK4) and 7 (MKK7) in embryonic stem cell (ESC) differentiation

Signal transduction pathways are integral components of the developmental regulatory network that guides progressive cell fate determination. MKK4 and MKK7 are upstream kinases of the mitogen-activated protein kinases (MAPKs), responsible for channeling physiological and environmental signals to their cellular responses. Both kinases are essential for survival of mouse embryos, but because of embryonic lethality, their precise developmental roles remain largely unknown. Using gene knock-out mouse ESCs, we studied the roles of MKK4 and MKK7 in differentiation in vitro. While MKK4 and MKK7 were dispensable for ESC self-renewal and pluripotency maintenance, they exhibited unique signaling and functional properties in differentiation. MKK4 and MKK7 complemented each other in activation of the JNK-c-Jun cascades and loss of both led to senescence upon cell differentiation. On the other hand, MKK4 and MKK7 had opposite effects on activation of the p38 cascades during differentiation. Specifically, MKK7 reduced p38 activation, while Mkk7(-/-) ESCs had elevated phosphorylation of MKK4, p38, and ATF2, and increased MEF2C expression. Consequently, Mkk7(-/-) ESCs had higher expression of MHC and MLC and enhanced formation of contractile cardiomyocytes. In contrast, MKK4 was required for p38 activation and Mkk4(-/-) ESCs exhibited diminished p-ATF2 and MEF2C expression, resulting in impaired MHC induction and defective cardiomyocyte differentiation. Exogenous MKK4 expression partially restored the ability of Mkk4(-/-) ESCs to differentiate into cardiomyocytes. Our results uncover complementary and interdependent roles of MKK4 and MKK7 in development, and identify the essential requirement for MKK4 in p38 activation and cardiomyocyte differentiation.     

Target of rapamycin complex 2 signals to downstream effector yeast protein kinase 2 (Ypk2) through adheres-voraciously-to-target-of-rapamycin-2 protein 1 (Avo1) in Saccharomyces cerevisiae

The conserved Ser/Thr kinase target of rapamycin (TOR) serves as a central regulator in controlling cell growth-related functions. There exist two distinct TOR complexes, TORC1 and TORC2, each coupling to specific downstream effectors and signaling pathways. In Saccharomyces cerevisiae, TORC2 is involved in regulating actin organization and maintaining cell wall integrity. Ypk2 (yeast protein kinase 2), a member of the cAMP-dependent, cGMP-dependent, and PKC (AGC) kinase family, is a TORC2 substrate known to participate in actin and cell wall regulation. Employing avo3(ts) mutants with defects in TORC2 functions that are suppressible by active Ypk2, we investigated the molecular interactions involved in mediating TORC2 signaling to Ypk2. GST pulldown assays in yeast lysates demonstrated physical interactions between Ypk2 and components of TORC2. In vitro binding assays revealed that Avo1 directly binds to Ypk2. In avo3(ts) mutants, the TORC2-Ypk2 interaction was reduced and could be restored by AVO1 overexpression, highlighting the important role of Avo1 in coupling TORC2 to Ypk2. The interaction was mapped to an internal region (amino acids 600-840) of Avo1 and a C-terminal region of Ypk2. Ypk2(334-677), a truncated form of Ypk2 containing the Avo1-interacting region, was able to interfere with Avo1-Ypk2 interaction in vitro. Overexpressing Ypk2(334-677) in yeast cells resulted in a perturbation of TORC2 functions, causing defective cell wall integrity, aberrant actin organization, and diminished TORC2-dependent Ypk2 phosphorylation evidenced by the loss of an electrophoretic mobility shift. Together, our data support the conclusion that the direct Avo1-Ypk2 interaction is crucial for TORC2 signaling to the downstream Ypk2 pathway.     

Sh3rf2/POSHER protein promotes cell survival by ring-mediated proteasomal degradation of the c-Jun N-terminal kinase scaffold POSH (Plenty of SH3s) protein

We report that Sh3rf2, a homologue of the pro-apoptotic scaffold POSH (Plenty of SH3s), acts as an anti-apoptotic regulator for the c-Jun N-terminal kinase (JNK) pathway. siRNA-mediated knockdown of Sh3rf2 promotes apoptosis of neuronal PC12 cells, cultured cortical neurons, and C6 glioma cells. This death appears to result from activation of JNK signaling. Loss of Sh3rf2 triggers activation of JNK and its target c-Jun. Also, apoptosis promoted by Sh3rf2 knockdown is inhibited by dominant-negative c-Jun as well as by a JNK inhibitor. Investigation of the mechanism by which Sh3rf2 regulates cell survival implicates POSH, a scaffold required for activation of pro-apoptotic JNK/c-Jun signaling. In cells lacking POSH, Sh3rf2 knockdown is unable to activate JNK. We further find that Sh3rf2 binds POSH to reduce its levels by a mechanism that requires the RING domains of both proteins and that appears to involve proteasomal POSH degradation. Conversely, knockdown of Sh3rf2 promotes the stabilization of POSH protein and activation of JNK signaling. Finally, we show that endogenous Sh3rf2 protein rapidly decreases following several different apoptotic stimuli and that knockdown of Sh3rf2 activates the pro-apoptotic JNK pathway in neuronal cells. These findings support a model in which Sh3rf2 promotes proteasomal degradation of pro-apoptotic POSH in healthy cells and in which apoptotic stimuli lead to rapid loss of Sh3rf2 expression, and consequently to stabilization of POSH and JNK activation and cell death. On the basis of these observations, we propose the alternative name POSHER (POSH-eliminating RING protein) for the Sh3rf2 protein.     

Receptor-type protein tyrosine phosphatase beta (RPTP-beta) directly dephosphorylates and regulates hepatocyte growth factor receptor (HGFR/Met) function

Protein tyrosine phosphorylation is a ubiquitous, fundamental biochemical mechanism that regulates essential eukaryotic cellular functions. The level of tyrosine phosphorylation of specific proteins is finely tuned by the dynamic balance between protein tyrosine kinase and protein tyrosine phosphatase activities. Hepatocyte growth factor receptor (also known as Met), a receptor protein tyrosine kinase, is a major regulator of proliferation, migration, and survival for many epithelial cell types. We report here that receptor-type protein tyrosine phosphatase β (RPTP-β) specifically dephosphorylates Met and thereby regulates its function. Expression of RPTP-β, but not other RPTP family members or catalytically inactive forms of RPTP-β, reduces hepatocyte growth factor (HGF)-stimulated Met tyrosine phosphorylation in HEK293 cells. Expression of RPTP-β in primary human keratinocytes reduces both basal and HGF-induced Met phosphorylation at tyrosine 1356 and inhibits downstream MEK1/2 and Erk activation. Furthermore, shRNA-mediated knockdown of endogenous RPTP-β increases basal and HGF-stimulated Met phosphorylation at tyrosine 1356 in primary human keratinocytes. Purified RPTP-β intracellular domain preferentially dephosphorylates purified Met at tyrosine 1356 in vitro. In addition, the substrate-trapping mutant of RPTP-β specifically interacts with Met in intact cells. Expression of RPTP-β in human primary keratinocytes reduces HGF induction of VEGF expression, proliferation, and motility. Taken together, the above data indicate that RPTP-β is a key regulator of Met function.     

Structural Mechanism for the Specific Assembly and Activation of the Extracellular Signal Regulated Kinase 5 (ERK5) Module

Mitogen-activated protein kinase (MAPK) activation depends on a linear binding motif found in all MAPK kinases (MKK). In addition, the PB1 (Phox and Bem1) domain of MKK5 is required for extracellular signal regulated kinase 5 (ERK5) activation. We present the crystal structure of ERK5 in complex with an MKK5 construct comprised of the PB1 domain and the linear binding motif. We show that ERK5 has distinct protein-protein interaction surfaces compared with ERK2, which is the closest ERK5 paralog. The two MAPKs have characteristically different physiological functions and their distinct protein-protein interaction surface topography enables them to bind different sets of activators and substrates. Structural and biochemical characterization revealed that the MKK5 PB1 domain cooperates with the MAPK binding linear motif to achieve substrate specific binding, and it also enables co-recruitment of the upstream activating enzyme and the downstream substrate into one signaling competent complex. Studies on present day MAPKs and MKKs hint on the way protein kinase networks may evolve. In particular, they suggest how paralogous enzymes with similar catalytic properties could acquire novel signaling roles by merely changing the way they make physical links to other proteins.     

Systematic Quantification of Negative Feedback Mechanisms in the Extracellular Signal-regulated Kinase (ERK) Signaling Network

Cell responses are actuated by tightly controlled signal transduction pathways. Although the concept of an integrated signaling network replete with interpathway cross-talk and feedback regulation is broadly appreciated, kinetic data of the type needed to characterize such interactions in conjunction with mathematical models are lacking. In mammalian cells, the Ras/ERK pathway controls cell proliferation and other responses stimulated by growth factors, and several cross-talk and feedback mechanisms affecting its activation have been identified. In this work, we take a systematic approach to parse the magnitudes of multiple regulatory mechanisms that attenuate ERK activation through canonical (Ras-dependent) and non-canonical (PI3K-dependent) pathways. In addition to regulation of receptor and ligand levels, we consider three layers of ERK-dependent feedback: desensitization of Ras activation, negative regulation of MEK kinase (e.g. Raf) activities, and up-regulation of dual-specificity ERK phosphatases. Our results establish the second of these as the dominant mode of ERK self-regulation in mouse fibroblasts. We further demonstrate that kinetic models of signaling networks, trained on a sufficient diversity of quantitative data, can be reasonably comprehensive, accurate, and predictive in the dynamical sense.     

Extracellular Signal-regulated Kinase 5 (ERK5) Mediates Prolactin-stimulated Adult Neurogenesis in the Subventricular Zone and Olfactory Bulb

Prolactin-stimulated adult neurogenesis in the subventricular zone (SVZ) and olfactory bulb (OB) mediates several reproductive behaviors including mating/pregnancy, dominant male pheromone preference in females, and paternal recognition of offspring. However, downstream signaling mechanisms underlying prolactin-induced adult neurogenesis are completely unknown. We report here for the first time that prolactin activates extracellular signal-regulated kinase 5 (ERK5), a MAP kinase that is specifically expressed in the neurogenic regions of the adult mouse brain. Knockdown of ERK5 by retroviral infection of shRNA attenuates prolactin-stimulated neurogenesis in SVZ-derived adult neural stem/progenitor cells (aNPCs). Inducible erk5 deletion in adult neural stem cells of transgenic mice inhibits neurogenesis in the SVZ and OB following prolactin infusion or mating/pregnancy. These results identify ERK5 as a novel and critical signaling mechanism underlying prolactin-induced adult neurogenesis.     

Phosphodiesterase 3A (PDE3A) deletion suppresses proliferation of cultured murine vascular smooth muscle cells (VSMCs) via inhibition of mitogen-activated protein kinase (MAPK) signaling and alterations in critical cell cycle regulatory proteins

Cyclic nucleotide phosphodiesterase 3 (PDE3) is an important regulator of cyclic adenosine monophosphate (cAMP) signaling within the cardiovascular system. In this study, we examined the role of PDE3A and PDE3B isoforms in regulation of growth of cultured vascular smooth muscle cells (VSMCs) and the mechanisms by which they may affect signaling pathways that mediate mitogen-induced VSMC proliferation. Serum- and PDGF-induced DNA synthesis in VSMCs grown from aortas of PDE3A-deficient (3A-KO) mice was markedly less than that in VSMCs from PDE3A wild type (3A-WT) and PDE3B-deficient (3B-KO) mice. The reduced growth response was accompanied by significantly less phosphorylation of extracellular signal-regulated kinase (ERK) in 3A-KO VSMCs, most likely due to a combination of greater site-specific inhibitory phosphorylation of Raf-1^Ser-259 by protein kinase A (PKA) and enhanced dephosphorylation of ERKs due to elevated mitogen-activated protein kinase phosphatase 1 (MKP-1). Furthermore, 3A-KO VSMCs, compared with 3A-WT, exhibited higher basal PKA activity and cAMP response element-binding protein (CREB) phosphorylation, higher levels of p53 and p53 phosphorylation, and elevated p21 protein together with lower levels of Cyclin-D1 and retinoblastoma (Rb) protein and Rb phosphorylation. Adenoviral overexpression of inactive CREB partially restored growth effects of serum in 3A-KO VSMCs. In contrast, exposure of 3A-WT VSMCs to VP16 CREB (active CREB) was associated with inhibition of serum-induced DNA synthesis similar to that in untreated 3A-KO VSMCs. Transfection of 3A-KO VSMCs with p53 siRNA reduced p21 and MKP-1 levels and completely restored growth without affecting amounts of Cyclin-D1 and Rb phosphorylation. We conclude that PDE3A regulates VSMC growth via two complementary pathways, i.e. PKA-catalyzed inhibitory phosphorylation of Raf-1 with resulting inhibition of MAPK signaling and PKA/CREB-mediated induction of p21, leading to G₀/G₁ cell cycle arrest, as well as by increased accumulation of p53, which induces MKP-1, p21, and WIP1, leading to inhibition of G₁ to S cell cycle progression.     

The Protein Factor-arrest 11 (Far11) Is Essential for the Toxicity of Human Caspase-10 in Yeast and Participates in the Regulation of Autophagy and the DNA Damage Signaling

The heterologous expression of human caspase-10 in Saccharomyces cerevisiae induces a lethal phenotype, which includes some hallmarks of apoptosis and autophagy, alterations in the intra-S checkpoint, and cell death. To determine the cellular processes and pathways that are responsible of the caspase-10-induced cell death we have designed a loss-of-function screening system to identify genes that are essential for the lethal phenotype. We observed that the ER-Golgi-localized family of proteins Far, MAPK signaling, the autophagy machinery, and several kinases and phosphatases are essential for caspase-10 toxicity. We also found that the expression of caspase-10 elicits a simultaneous activation of the MAP kinases Fus3, Kss1, and Slt2. Furthermore, the protein Far11, which is a target of MAP kinases, is essential for the dephosphorylation of Atg13 and, consequently, for the induction of autophagy. In addition, Far11 participates in the regulation of the DNA damage response through the dephosphorylation of Rad53. Finally, we have also demonstrated that Far11 is able to physically interact with the phosphatases Pph21, Pph22, and Pph3. Overall, our results indicate that the expression of human caspase-10 in S. cerevisiae activates an intracellular death signal that depends on the Far protein complex and that Far11 may function as a regulator subunit of phosphatases in different processes, thus representing a mechanistic link between them.     

Involvement of polo-like kinase 1 (Plk1) in mitotic arrest by inhibition of mitogen-activated protein kinase-extracellular signal-regulated kinase-ribosomal S6 kinase 1 (MEK-ERK-RSK1) cascade

Cell division is controlled through cooperation of different kinases. Of these, polo-like kinase 1 (Plk1) and p90 ribosomal S6 kinase 1 (RSK1) play key roles. Plk1 acts as a G(2)/M trigger, and RSK1 promotes G(1) progression. Although previous reports show that Plk1 is suppressed by RSK1 during meiosis in Xenopus oocytes, it is still not clear whether this is the case during mitosis or whether Plk1 counteracts the effects of RSK1. Few animal models are available for the study of controlled and transient cell cycle arrest. Here we show that encysted embryos (cysts) of the primitive crustacean Artemia are ideal for such research because they undergo complete cell cycle arrest when they enter diapause (a state of obligate dormancy). We found that Plk1 suppressed the activity of RSK1 during embryonic mitosis and that Plk1 was inhibited during embryonic diapause and mitotic arrest. In addition, studies on HeLa cells using Plk1 siRNA interference and overexpression showed that phosphorylation of RSK1 increased upon interference and decreased after overexpression, suggesting that Plk1 inhibits RSK1. Taken together, these findings provide insights into the regulation of Plk1 during cell division and Artemia diapause cyst formation and the correlation between the activity of Plk1 and RSK1.     

Sprouty-related Ena/vasodilator-stimulated phosphoprotein homology 1-domain-containing protein (SPRED1), a tyrosine-protein phosphatase non-receptor type 11 (SHP2) substrate in the Ras/extracellular signal-regulated kinase (ERK) pathway

SHP2 is a tyrosine phosphatase involved in the activation of the Ras/ERK signaling pathway downstream of a number of receptor tyrosine kinases. One of the proposed mechanisms involving SHP2 in this context is to dephosphorylate and inactivate inhibitors of the Ras/ERK pathway. Two protein families bearing a unique, common domain, Sprouty and SPRED proteins, are possible candidates because they have been reported to inhibit the Ras/ERK pathway upon FGF activation. We tested whether any of these proteins are likely substrates of SHP2. Our findings indicate that Sprouty2 binds to the C-terminal tail of SHP2, which is an unlikely substrate binding site, whereas SPRED proteins bind to the tyrosine phosphatase domain that is known to be the binding site for its substrates. Overexpressed SHP2 was able to dephosphorylate SPREDs but not Sprouty2. Finally, we found two tyrosine residues on SPRED1 that are required, when phosphorylated, to inhibit Ras/ERK activation and identified Tyr-420 as a specific dephosphorylation target of SHP2. The evidence obtained indicates that SPRED1 is a likely substrate of SHP2, whose tyrosine dephosphorylation is required to attenuate the inhibitory action of SPRED1 in the Ras/ERK pathway.     

Association of the Breast Cancer Antiestrogen Resistance Protein 1 (BCAR1) and BCAR3 Scaffolding Proteins in Cell Signaling and Antiestrogen Resistance

Most breast cancers are estrogen receptor-positive and treated with antiestrogens, but aberrant signaling networks can induce drug resistance. One of these networks involves the scaffolding protein BCAR1/p130CAS, which regulates cell growth and migration/invasion. A less investigated scaffolding protein that also confers antiestrogen resistance is the SH2 domain-containing protein BCAR3. BCAR1 and BCAR3 bind tightly to each other through their C-terminal domains, thus potentially connecting their associated signaling networks. However, recent studies using BCAR1 and BCAR3 interaction mutants concluded that association between the two proteins is not critical for many of their interrelated activities regulating breast cancer malignancy. We report that these previously used BCAR mutations fail to cause adequate loss-of-function of the complex. By using structure-based BCAR1 and BCAR3 mutants that lack the ability to interact, we show that BCAR3-induced antiestrogen resistance in MCF7 breast cancer cells critically depends on its ability to bind BCAR1. Interaction with BCAR3 increases the levels of phosphorylated BCAR1, ultimately potentiating BCAR1-dependent antiestrogen resistance. Furthermore, antiestrogen resistance in cells overexpressing BCAR1/BCAR3 correlates with increased ERK1/2 activity. Inhibiting ERK1/2 through overexpression of the regulatory protein PEA15 negates the resistance, revealing a key role for ERK1/2 in BCAR1/BCAR3-induced antiestrogen resistance. Reverse-phase protein array data show that PEA15 levels in invasive breast cancers correlate with patient survival, suggesting that PEA15 can override ERK1/2 activation by BCAR1/BCAR3 and other upstream regulators. We further uncovered that the BCAR3-related NSP3 can also promote antiestrogen resistance. Thus, strategies to disrupt BCAR1-BCAR3/NSP3 complexes and associated signaling networks could ultimately lead to new breast cancer therapies.