In the beginning, there was protein phosphorylation.

The importance of reversible protein phosphorylation to cellular regulation cannot be overstated. In eukaryotic cells, protein kinase/phosphatase signaling pathways regulate a staggering number of cellular processes, including cell proliferation, cell death (apoptosis, necroptosis, necrosis), metabolism (at both the cellular and organismal levels), behavior and neurological function, development, and pathogen resistance. Although protein phosphorylation as a mode of eukaryotic cell regulation is familiar to most biochemists, many are less familiar with protein kinase/phosphatase signaling networks that function in prokaryotes. In this thematic minireview series, we present four minireviews that cover the important field of prokaryotic protein phosphorylation.

Glycogen synthase kinase-3ß inhibition ameliorates cardiac parasympathetic dysfunction in type 1 diabetic Akita mice.

Decreased heart rate variability (HRV) is a major risk factor for sudden death and cardiovascular disease. We previously demonstrated that parasympathetic dysfunction in the heart of the Akita type 1 diabetic mouse was due to a decrease in the level of the sterol response element-binding protein (SREBP-1). Here we demonstrate that hyperactivity of glycogen synthase kinase-3ß (GSK3ß) in the atrium of the Akita mouse results in decreased SREBP-1, attenuation of parasympathetic modulation of heart rate, measured as a decrease in the high-frequency (HF) fraction of HRV in the presence of propranolol, and a decrease in expression of the G-protein coupled inward rectifying K(+) (GIRK4) subunit of the acetylcholine (ACh)-activated inward-rectifying K(+) channel (IKACh), the ion channel that mediates the heart rate response to parasympathetic stimulation. Treatment of atrial myocytes with the GSK3ß inhibitor Kenpaullone increased levels of SREBP-1 and expression of GIRK4 and IKACh, whereas a dominant-active GSK3ß mutant decreased SREBP-1 and GIRK4 expression. In Akita mice treated with GSK3ß inhibitors Li(+) and/or CHIR-99021, Li(+) increased IKACh, and Li(+) and CHIR-99021 both partially reversed the decrease in HF fraction while increasing GIRK4 and SREBP-1 expression. These data support the conclusion that increased GSK3ß activity in the type 1 diabetic heart plays a critical role in parasympathetic dysfunction through an effect on SREBP-1, supporting GSK3ß as a new therapeutic target for diabetic autonomic neuropathy.

Mammalian MAPK signal transduction pathways activated by stress and inflammation: a 10-year update.

The mammalian stress-activated families of mitogen-activated protein kinases (MAPKs) were first elucidated in 1994, and by 2001, substantial progress had been made in identifying the architecture of the pathways upstream of these kinases as well as in cataloguing candidate substrates. This information remains largely sound. Nevertheless, an informed understanding of the physiological and pathophysiological roles of these kinases remained to be accomplished. In the past decade, there has been an explosion of new work using RNAi in cells, as well as transgenic, knockout and conditional knockout technology in mice that has provided valuable insight into the functions of stress-activated MAPK pathways. These findings have important implications in our understanding of organ development, innate and acquired immunity, and diseases such as atherosclerosis, tumorigenesis, and type 2 diabetes. These new developments bring us within striking distance of the development and validation of novel treatment strategies. Herein we first summarize the molecular components of the mammalian stress-regulated MAPK pathways and their regulation as described thus far. We then review some of the in vivo functions of these pathways.

Decreased metalloprotease 9 induction, cardiac fibrosis, and higher autophagy after pressure overload in mice lacking the transcriptional regulator p8.

Left ventricular remodeling, including the deposition of excess extracellular matrix, is key to the pathogenesis of heart failure. The stress-inducible transcriptional regulator p8 is increased in failing human hearts and is required both for agonist-stimulated cardiomyocyte hypertrophy and for cardiac fibroblasts matrix metalloprotease-9 (MMP9) induction. In the heart, upregulation of autophagy is an adaptive response to stress and plays a causative role in cardiomyopathies. We have recently shown that p8 ablation in cardiac cells upregulates autophagy and that, in vivo, loss of p8 results in a decrease of cardiac function. Here we investigated the effects of p8 genetic deletion in mediating adverse myocardial remodeling. Unstressed p8-/- mouse hearts manifested complex alterations in the expression of fibrosis markers. In addition, these mice displayed elevated autophagy and apoptosis compared with p8+/+ mice. Transverse aortic constriction (TAC) induced left ventricular p8 expression in p8+/+ mice. Pressure overload caused left ventricular remodeling in both genotypes, however, p8-/- mice showed less cardiac fibrosis induction. Consistent with this, although MMP9 induction was attenuated in the p8-/- mice, induction of MMP2 and MMP3 were strikingly upregulated while TIMP2 was downregulated. Left ventricular autophagy increased after TAC and was significantly higher in the p8-/- mice. Thus p8-deletion results in reduced collagen fibrosis after TAC, but in turn, is associated with a detrimental higher increase in autophagy. These findings suggest a role for p8 in regulating in vivo key signaling pathways involved in the pathogenesis of heart failure.

Activation of SAPK/JNKs in vitro.

The stress-activated protein kinase/c-jun N-terminal kinases (SAPK/JNKs) are mitogen-activated protein kinases (MAPKs) that are activated by stressful and inflammatory stimuli and regulate cellular responses such as proliferation, differentiation, and apoptosis. The SAPK/JNKs are phosphorylated and activated by the MAP kinase kinases (MAP2Ks), SEK1/MKK4 and MKK7. These MAP2Ks are phosphorylated and activated by upstream stress-activated MAPK kinase kinases (MAP3Ks). Upon activation, SAPK/JNKs translocate to the nucleus and phosphorylate transcription factors, ultimately resulting in the modulation of gene expression. We have analyzed the activation of SAPK/JNK and stress-activated MAP3Ks using in vitro kinase assays. In addition, we have studied the role of different MAP3Ks in SAPK/JNK signaling by silencing specific MAP3K expression with RNAi and then analyzing the effect on activation of SAPK/JNKs and other MAPKs.

Mammalian Ste20-like kinase (Mst2) indirectly supports Raf-1/ERK pathway activity via maintenance of protein phosphatase-2A catalytic subunit levels and consequent suppression of inhibitory Raf-1 phosphorylation.

Many tumor suppressor proteins act to blunt the effects of mitogenic signaling pathways. Loss of function mutations in the merlin tumor suppressor underlie neurofibromatosis type 2 (NF2), a familial autosomal dominant cancer syndrome. Studies of Drosophila suggest that Hippo (hpo) is required for inhibition of cell proliferation mediated by dMer, the orthologue of human merlin. Mammalian sterile 20-like kinase-2 (Mst2) is a mammalian Hpo orthologue, and numerous studies implicate Mst2 as a tumor suppressor. Mst2 is negatively regulated by the proto-oncoprotein Raf-1 in a manner independent of the kinase activity of Raf-1. We sought to determine whether, in mammalian cells, merlin could positively regulate Mst2. We also sought to determine whether Mst2, in addition to being negatively regulated by Raf-1, might itself reciprocally regulate Raf-1. In contrast to findings from Drosophila, we find no evidence that mammalian merlin positively regulates mammalian Mst2. Instead, surprisingly, RNA interference silencing of Mst2 leads to elevated inhibitory phosphorylation of Raf-1 at Ser-259 and impaired Raf-1 kinase activity. Consequent to this, ERK pathway activation and cell proliferation are attenuated. Phosphatase-2A (PP2A) dephosphorylates Raf-1 Ser-259 in response to mitogens. Interestingly RNA interference silencing of Mst2 triggers a striking proteasome-dependent decrease in the levels of the catalytic subunit of PP2A (PP2A-C). A similar effect is achieved upon silencing of large tumor suppressor (LATS)-1 and LATS2, direct substrates of Mst2. Our studies reveal a more complex role for Mst2 than previously thought. The Mst2 --> LATS1/2 pathway, by maintaining PP2A-C levels, may, in some situations, positively affect mitogenic signaling.

Deficiency of the transcriptional regulator p8 results in increased autophagy and apoptosis, and causes impaired heart function.

Autophagy is a cytoprotective pathway used to degrade and recycle cytoplasmic content. Dysfunctional autophagy has been linked to both cancer and cardiomyopathies. Here, we show a role for the transcriptional regulator p8 in autophagy. p8 RNA interference (RNAi) increases basal autophagy markers in primary cardiomyocytes, in H9C2 and U2OS cells, and decreases cellular viability after autophagy induction. This autophagy is associated with caspase activation and is blocked by atg5 silencing and by pharmacological inhibitors. FoxO3 transcription factor was reported to activate autophagy by enhancing the expression of autophagy-related genes. P8 expression represses FoxO3 transcriptional activity, and p8 knockdown affects FoxO3 nuclear localization. Thus, p8 RNAi increases FoxO3 association with bnip3 promoter, a known proautophagic FoxO3 target, resulting in higher bnip3 RNA and protein levels. Accordingly, bnip3 knockdown restores cell viability and blocks apoptosis of p8-deficient cells. In vivo, p8 -/- mice have higher autophagy and express higher cardiac bnip3 levels. These mice develop left ventricular wall thinning and chamber dilation, with consequent impaired cardiac function. Our studies provide evidence of a p8-dependent mechanism regulating autophagy by acting as FoxO3 corepressor, which may be relevant for diseases associated with dysregulated autophagy, as cardiovascular pathologies and cancer.

Deficiency of transcriptional regulator p8 induces autophagy and causes impaired cardiac function.

Through autophagy cells adapt to nutrient availability, recycle cellular material and eliminate toxic proteins and damaged cellular organelles. Dysregulation of autophagy is implicated in the pathogenesis of various diseases, including cancer, neurodegeneration and cardiomyopathies. The transcription factor FoxO3 activates autophagy by enhancing the expression of several genes. We find a role for the transcriptional regulator p8 in controling autophagy by repressing FoxO3 transcriptional activity. p8 silencing increases the association of FoxO3 with the bnip3 promoter, a known pro-autophagic FoxO3 target, and results in increasead basal autophagy and decreased cellular viability. Likewise, p8 overexpression inhibits Bnip3 upregulation after autophagy activation. Thus, p8 appears to antagonize the promotion of autophagy mediated by the FoxO3-Bnip3 axis. Consistent with this, bnip3 knockdown restores viability in p8-deficient cells. In vivo, hearts from p8-/- mice have higher basal autophagy and bnip3 levels. These mice develop left ventricular wall thinning and chamber dilation, with consequent impaired cardiac function.

GCK is essential to systemic inflammation and pattern recognition receptor signaling to JNK and p38.

Systemic inflammation arising from the organismal distribution of pathogen-associated molecular patterns is a major cause of clinical morbidity and mortality. Herein we report a critical and previously unrecognized in vivo role for germinal center kinase (GCK, genome nomenclature: map4k2), a mammalian Sterile 20 (STE20) orthologue, in PAMP signaling, and systemic inflammation. We find that disruption of gck in mice strongly impairs PAMP-stimulated macrophage cytokine and chemokine release and renders mice resistant to endotoxin-mediated lethality. Bone marrow transplantation studies show that hematopoietic cell GCK signaling is essential to systemic inflammation. Disruption of gck substantially reduces PAMP activation of macrophage Jun-N-terminal kinase (JNK) and p38 mitogen-activated protein kinases (MAPKs) via reduced activation of the MAPK-kinase-kinases (MAP3Ks) mixed lineage kinases (MLKs)-2 and -3. Extracellular signal-regulated kinase (ERK) and nuclear factor-kappaB (NF-kappaB) activation are largely unaffected. Thus, GCK is an essential PAMP effector coupling JNK and p38, but not ERK or NF-kappaB to systemic inflammation.

SOK1 translocates from the Golgi to the nucleus upon chemical anoxia and induces apoptotic cell death.

SOK1 is a Ste20 protein kinase of the germinal center kinase (GCK) family that has been shown to be activated by oxidant stress and chemical anoxia, a cell culture model of ischemia. More recently, it has been shown to be localized to the Golgi apparatus, where it functions in a signaling pathway required for cell migration and polarization. Herein, we demonstrate that SOK1 regulates cell death after chemical anoxia, as its down-regulation by RNA interference enhances cell survival. Furthermore, expression of SOK1 elicits apoptotic cell death by activating the intrinsic pathway. We also find that a cleaved form of SOK1 translocates from the Golgi to the nucleus after chemical anoxia and that this translocation is dependent on both caspase activity and on amino acids 275-292, located immediately C-terminal to the SOK1 kinase domain. Furthermore, SOK1 entry into the nucleus is important for the cell death response since SOK1 mutants unable to enter the nucleus do not induce cell death. In summary, SOK1 is necessary to induce cell death and can induce death when overexpressed. Furthermore, SOK1 appears to play distinctly different roles in stressed versus non-stressed cells, regulating cell death in the former.

Dissection of a signaling pathway by which pathogen-associated molecular patterns recruit the JNK and p38 MAPKs and trigger cytokine release.

Pathogen-associated molecular patterns (PAMPs), molecular moieties produced by invading microbial pathogens, initiate innate immune responses by binding to pattern recognition receptors (PRRs). Engagement of PRRs elicits a wide variety of responses, including the production and release of cytokines and chemokines. These responses require the activation of several parallel signaling pathways, including NF-kappaB, the interferon regulatory factors, and the MAPKs. The JNK and p38 MAPK groups are major PRR effectors and are key to the PRR-dependent induction and release of proinflammatory cytokines such as tumor necrosis factor and interleukin-8. The mammalian Ste20 orthologue germinal center kinase (GCK) is required for the activation of JNK by a subset of PAMPs; however, the mechanisms by which GCK couples to downstream events remain unclear. Here we show that GCK is required for JNK and, unexpectedly, p38 activation by three bacterial PAMPs, lipopolysaccharide, peptidoglycan, and flagellin (FliC). We show that these same PAMPs, in a GCK-dependent manner, activate mixed lineage kinases-2 and -3, MAPK kinase kinases upstream of JNK, and p38. We also show that MLK2 and -3 are required for activation of JNK and p38 by ectopically expressed GCK. Finally, we show that MLK2 and -3 are required for lipopolysaccharide, peptidoglycan, and FliC recruitment of JNK and p38 as well as for PAMP recruitment of the transcription factor c-Jun, and for the induction of interleukin-8. Our results define a signaling pathway whereby PAMPs can trigger MAPK activation and gene expression.

The integration of signaling by multiprotein complexes containing Raf kinases.

In vivo, eukaryotic cells are subjected simultaneously to a broad array of signals ranging from mitogens and inflammatory inputs to environmental stresses and developmental cues. The combinatorial nature of cellular signaling necessitates that a cell integrate its signal transduction pathways so as to implement rapidly and efficiently an appropriate suite of responses. Emerging evidence indicates that, over the course of evolution, cells have developed multiprotein signaling complexes, or "signalosomes" that mediate the coordinate regulation of different signaling pathways. Such molecular signal integration contrasts with the classical notion of signaling complexes assembled by scaffold proteins-entities that function to segregate specific pathways from one another. This review will focus on two signal integrating multiprotein complexes that involve Raf family kinases: the MLK3-B-Raf-Raf-1 complex and the Raf-1-Mst-2 complex.

The GCK II and III subfamilies of the STE20 group kinases.

The Ste20 (sterile 20) proteins are a large family of serine/threonine kinases. Since their discovery a growing body of evidence has implicated them in the regulation of signaling pathways governing cell growth, cell differentiation cell death and cell volume. Approximately 30 human members have been identified based on the high degree of homology of their catalytic domain to that of the Ste20p from Saccharomyces cerevisiae. In addition to the conserved regions, there are also regions of sequence that make each of them unique. In this review we will focus on two subfamilies of the group, GCK-II and GCK-III, families that are closely related but, again, unique in their structural features and biological functions. Herein, we will present what we hope will be the current state of knowledge about these kinases, and discuss what remains to be done in order to better understand their activity and regulation.

Helix-loop-helix protein p8, a transcriptional regulator required for cardiomyocyte hypertrophy and cardiac fibroblast matrix metalloprotease induction.

Cardiomyocyte hypertrophy and extracellular matrix remodeling, primarily mediated by inflammatory cytokine-stimulated cardiac fibroblasts, are critical cellular events in cardiac pathology. The molecular components governing these processes remain nebulous, and few genes have been linked to both hypertrophy and matrix remodeling. Here we show that p8, a small stress-inducible basic helix-loop-helix protein, is required for endothelin- and alpha-adrenergic agonist-induced cardiomyocyte hypertrophy and for tumor necrosis factor-stimulated induction, in cardiac fibroblasts, of matrix metalloproteases (MMPs) 9 and 13-MMPs linked to general inflammation and to adverse ventricular remodeling in heart failure. In a stimulus-dependent manner, p8 associates with chromatin containing c-Jun and with the cardiomyocyte atrial natriuretic factor (anf) promoter and the cardiac fibroblast mmp9 and mmp13 promoters, established activator protein 1 effectors. p8 is also induced strongly in the failing human heart by a process reversed upon therapeutic intervention. Our results identify an unexpectedly broad involvement for p8 in key cellular events linked to cardiomyocyte hypertrophy and cardiac fibroblast MMP production, both of which occur in heart failure.

Gene 33/RALT is induced by hypoxia in cardiomyocytes, where it promotes cell death by suppressing phosphatidylinositol 3-kinase and extracellular signal-regulated kinase survival signaling.

Ischemia in the heart deprives cardiomyocytes of oxygen, triggering cell death (myocardial infarction). Ischemia and its cell culture model, hypoxia, elicit a stress response program that contributes to cardiomyocyte death; however, the molecular components required to promote this process remain nebulous. Gene 33 is a 50-kDa cytosolic adapter protein that suppresses signaling from receptor Tyr kinases of the epidermal growth factor receptor/ErbB family. Here we show that adenoviral expression of Gene 33 swiftly stimulates cardiomyocyte death coincident with reduced Akt and extracellular signal-regulated kinase (ERK) signaling. Subjecting cardiomyocytes to hypoxia and then reoxygenation induces gene 33 mRNA and Gene 33 protein. RNA interference experiments indicate that endogenous Gene 33 reduces Akt and ERK signaling and is required for maximal hypoxia-induced cardiomyocyte death. Gene 33 levels are also strikingly increased in myocardial ischemic injury and infarction. Our results identify a new role for Gene 33 as a component in the molecular pathophysiology of ischemic injury.

Mixed-lineage kinase 3 regulates B-Raf through maintenance of the B-Raf/Raf-1 complex and inhibition by the NF2 tumor suppressor protein.

The Ras --> Raf --> MEK1/2 --> extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway couples mitogenic signals to cell proliferation. B-Raf and Raf-1 function within an oligomer wherein they are regulated in part by mutual transactivation. The MAPK kinase kinase (MAP3K) mixed-lineage kinase 3 (MLK3) is required for mitogen activation of B-Raf and cell proliferation. Here we show that the kinase activity of MLK3 is not required for support of B-Raf activation. Instead, MLK3 is a component of the B-Raf/Raf-1 complex and is required for maintenance of the integrity of this complex. We show that the activation of ERK and the proliferation of human schwannoma cells bearing a loss-of-function mutation in the neurofibromatosis 2 (NF2) gene require MLK3. We find that merlin, the product of NF2, blunts the activation of both ERK and c-Jun N-terminal kinase (JNK). Finally, we demonstrate that merlin and MLK3 can interact in situ and that merlin can disrupt the interactions between B-Raf and Raf-1 or those between MLK3 and either B-Raf or Raf-1. Thus, MLK3 is part of a multiprotein complex and is required for ERK activation. The levels of this complex may be negatively regulated by merlin.

c-Jun N-terminal kinases mediate reactivation of Akt and cardiomyocyte survival after hypoxic injury in vitro and in vivo.

Akt is a central regulator of cardiomyocyte survival after ischemic injury in vitro and in vivo, but the mechanisms regulating Akt activity in the postischemic cardiomyocyte are not known. Furthermore, although much is known about the detrimental role that the c-Jun N-terminal kinases (JNKs) play in promoting death of cells exposed to various stresses, little is known of the molecular mechanisms by which JNK activation can be protective. We report that JNKs are necessary for the reactivation of Akt after ischemic injury. We identified Thr450 of Akt as a residue that is phosphorylated by JNKs, and the phosphorylation status of Thr450 regulates reactivation of Akt after hypoxia, apparently by priming Akt for subsequent phosphorylation by 3-phosphoinositide-dependent protein kinase. The reduction in Akt activity that is induced by JNK inhibition may have significant biological consequences, as we find that JNKs, acting via Akt, are critical determinants of survival in posthypoxic cardiomyocytes in culture. Furthermore, in contrast to selective p38-mitogen-activated protein kinase inhibition, which was cardioprotective in vivo, concurrent inhibition of both JNKs and p38-mitogen-activated protein kinases increased ischemia/reperfusion injury in the heart of the intact rat. These studies demonstrate that reactivation of Akt after resolution of hypoxia and ischemia is regulated by JNKs and suggest that this is likely a central mechanism of the myocyte protective effect of JNKs.

Gene 33 is an endogenous inhibitor of epidermal growth factor (EGF) receptor signaling and mediates dexamethasone-induced suppression of EGF function.

We report a mechanism by which the adapter protein Gene 33 (also called RALT and MIG6) regulates epidermal growth factor receptor (EGFR) signaling. We find that Gene 33 inhibits EGFR autophosphorylation and specifically blunts epidermal growth factor (EGF)-induced activation and/or phosphorylation of Ras, ERK, JNK, Akt/PKB, and retinoblastoma protein. The Ack homology domain of Gene 33, which contains the previously identified EGFR binding domain, is both necessary and sufficient for this inhibition of EGFR autophosphorylation. The endogenous Gene 33 polypeptide is induced by EGF, platelet-derived growth factor, serum, and dexamethasone (Dex) in Rat 2 rat fibroblasts. Dex induces Gene 33 expression and inhibits EGFR phosphorylation and EGF signaling. RNA interference-mediated silencing of Gene 33 significantly reverses this effect. Overexpression of Gene 33 completely blocks EGF-induced protein and DNA synthesis in Rat 2 cells, whereas gene 33 RNA interference substantially enhances EGF-induced protein and DNA synthesis in Rat 2 cells. Our results indicate that Gene 33 is a physiological feedback inhibitor of the EGFR, functioning to inhibit EGFR phosphorylation and all events induced by EGFR activation. Our results also indicate a role for Gene 33 in the suppression, by Dex, of EGF signaling pathways. We propose that Gene 33 may function in the cross-talk between EGF signaling and other mitogenic and/or stress signaling pathways.