Anthony J. Muslin

Interaction of 14-3-3 with signaling proteins is mediated by the recognition of phosphoserine.

The highly conserved and ubiquitously expressed 14-3-3 family of proteins bind to a variety of proteins involved in signal transduction and cell cycle regulation. The nature and specificity of 14-3-3 binding is, however, not known. Here we show that 14-3-3 is a specific phosphoserine-binding protein. Using a panel of phosphorylated peptides based on Raf-1, we have defined the 14-3-3 binding motif and show that most of the known 14-3-3 binding proteins contain the motif. Peptides containing the motif could disrupt 14-3-3 complexes and inhibit maturation of Xenopus laevis oocytes. These results suggest that the interactions of 14-3-3 with signaling proteins are critical for the activation of signaling proteins. Our findings also suggest novel roles for serine/threonine phosphorylation in the assembly of protein-protein complexes.

Akt1 Is Required for Physiological Cardiac Growth

Background— Postnatal growth of the heart chiefly involves nonproliferative cardiomyocyte enlargement. Cardiac hypertrophy exists in a “physiological” form that is an adaptive response to long-term exercise training and as a “pathological” form that often is a maladaptive response to provocative stimuli such as hypertension and aortic valvular stenosis. A signaling cascade that includes the protein kinase Akt regulates the growth and survival of many cell types, but the precise role of Akt1 in either form of cardiac hypertrophy is unknown. Methods and Results— To evaluate the role of Akt1 in physiological cardiac growth, akt1−/− adult murine cardiac myocytes (AMCMs) were treated with IGF-1, and akt1−/− mice were subjected to exercise training. akt1−/− AMCMs were resistant to insulin-like growth factor-1–stimulated protein synthesis. The akt1−/− mice were found to be resistant to swimming training–induced cardiac hypertrophy. To evaluate the role of Akt in pathological cardiac growth, akt1−/− AMCMs were treated with endothelin-1, and akt1−/− mice were subjected to pressure overload by transverse aortic constriction. Surprisingly, akt1−/− AMCMs were sensitized to endothelin-1–induced protein synthesis, and akt1−/− mice developed an exacerbated form of cardiac hypertrophy in response to transverse aortic constriction. Conclusions— These results establish Akt1 as a pivotal regulatory switch that promotes physiological cardiac hypertrophy while antagonizing pathological hypertrophy.

Hypertension and prolonged vasoconstrictor signaling in RGS2-deficient mice

Signaling by hormones and neurotransmitters that activate G protein-coupled receptors (GPCRs) maintains blood pressure within the normal range despite large changes in cardiac output that can occur within seconds. This implies that blood pressure regulation requires precise kinetic control of GPCR signaling. To test this hypothesis, we analyzed mice deficient in RGS2, a GTPase-activating protein that greatly accelerates the deactivation rate of heterotrimeric G proteins in vitro. Both rgs2+/- and rgs2-/- mice exhibited a strong hypertensive phenotype, renovascular abnormalities, persistent constriction of the resistance vasculature, and prolonged response of the vasculature to vasoconstrictors in vivo. Analysis of P2Y receptor-mediated Ca2+ signaling in vascular smooth muscle cells in vitro indicated that loss of RGS2 increased agonist potency and efficacy and slowed the kinetics of signal termination. These results establish that abnormally prolonged signaling by G protein-coupled vasoconstrictor receptors can contribute to the onset of hypertension, and they suggest that genetic defects affecting the function or expression of RGS2 may be novel risk factors for development of hypertension in humans.

14-3-3 proteins block apoptosis and differentially regulate MAPK cascades

14‐3‐3 family members are dimeric phosphoserine‐binding proteins that participate in signal transduction and checkpoint control pathways. In this work, dominant‐negative mutant forms of 14‐3‐3 were used to disrupt 14‐3‐3 function in cultured cells and in transgenic animals. Transfection of cultured fibroblasts with the R56A and R60A double mutant form of 14‐3‐3ζ (DN‐14‐3‐3ζ) inhibited serum‐stimulated ERK MAPK activation, but increased the basal activation of JNK1 and p38 MAPK. Fibroblasts transfected with DN‐14‐3‐3ζ exhibited markedly increased apoptosis in response to UVC irradiation that was blocked by pre‐treatment with a p38 MAPK inhibitor, SB202190. Targeted expression of DN‐14‐3‐3η to murine postnatal cardiac tissue increased the basal activation of JNK1 and p38 MAPK, and affected the ability of mice to compensate for pressure overload, which resulted in increased mortality, dilated cardiomyopathy and massive cardiomyocyte apoptosis. These results demonstrate that a primary function of mammalian 14‐3‐3 proteins is to inhibit apoptosis.

MAPK signalling in cardiovascular health and disease: molecular mechanisms and therapeutic targets.

Intracellular MAPK (mitogen-activated protein kinase) signalling cascades probably play an important role in the pathogenesis of cardiac and vascular disease. A substantial amount of basic science research has defined many of the details of MAPK pathway organization and activation, but the role of individual signalling proteins in the pathogenesis of various cardiovascular diseases is still being elucidated. In the present review, the role of the MAPKs ERK (extracellular signal-regulated kinase), JNK (c-Jun N-terminal kinase) and p38 MAPK in cardiac hypertrophy, cardiac remodelling after myocardial infarction, atherosclerosis and vascular restenosis will be examined, with attention paid to genetically modified murine model systems and to the use of pharmacological inhibitors of protein kinases. Despite the complexities of this field of research, attractive targets for pharmacological therapy are emerging.

The protein kinase KSR interacts with 14-3-3 protein and Raf

BACKGROUND KSR (kinase suppressor of Ras) is a recently identified putative protein kinase that positively mediates the Ras signaling pathway in the invertebrates Caenorhabditis elegans and Drosophila melanogaster. The function of vertebrate KSR is not well characterized biochemically or biologically. RESULTS We examined the physiological role of KSR in vertebrate signal transduction using Xenopus laevis oocytes. Overexpression of KSR, in combination with overexpression of the intracellular dimeric protein 14-3-3, induced Xenopus oocyte meiotic maturation and cdc2 kinase activation; the effect of KSR and 14-3-3 on oocyte maturation was blocked by co-expression of dominant-negative Raf-1. We noted that KSR contains multiple potential binding sites for 14-3-3, and we used the yeast two-hybrid system and co-immunoprecipitation experiments to show that KSR can bind to 14-3-3. Furthermore, we demonstrated that KSR can form a complex with Raf kinase both in vitro and in cultured cells. Cell fractionation studies revealed that KSR formed a complex with 14-3-3 in both the membrane and cytoplasmic fractions of cell lysates; however, KSR only formed a complex with Raf-1 in the membrane fraction. CONCLUSIONS Our finding suggest that KSR, 14-3-3 and Raf form an oligomeric signaling complex and that KSR positively regulates the Ras signaling pathway in vertebrate organisms.

Raf-1 kinase is essential for early Xenopus development and mediates the induction of mesoderm by FGF

Animal cap explants from Xenopus embryos injected with a dominant negative Raf-1 mutant, termed NAF (not a functional Raf), demonstrated a complete block to basic fibroblast growth factor (FGF)-stimulated mesoderm induction. Activin induction of mesoderm was normal in embryos that expressed NAF. Injection of NAF RNA into 2-cell stage embryos blocked normal development during neurula stages and caused severe posterior truncations in tadpoles. The phenotype induced by NAF could be rescued by coinjection of wild-type raf-1 RNA. The NAF mutant functioned by specifically blocking the activation of endogenous Raf kinase activity. These findings suggest that Raf-1 mediates FGF, but not activin, receptor signaling during mesoderm induction and implicate Raf-1 as a key signaling molecule in the development of posterior structure.

Raf-1 Kinase Is Required for Cardiac Hypertrophy and Cardiomyocyte Survival in Response to Pressure Overload

Background—Cardiac hypertrophy is a common response to pressure overload and is associated with increased mortality. Mechanical stress in the heart results in the activation of the small GTPase ras and the Raf-1/MEK/ERK signaling cascade in addition to other signaling pathways. Methods and Results—In an attempt to determine the requirement for the serine/threonine kinase Raf-1 in the pathogenesis of cardiac hypertrophy, we generated transgenic mice with cardiac-specific expression of a dominant negative form of Raf-1 (DN-Raf). DN-Raf mice appeared normal at birth, were fertile, and had normal cardiac structure and function in the absence of provocative stimulation. In response to pressure overload, cardiac extracellular signal-regulated kinase (ERK) activation was inhibited, but c-Jun N-terminal kinase (JNK) activation and p38 mitogen-activated protein kinase (MAPK) activation were normal. DN-Raf mice were sensitized to pressure overload and the development of cardiomyocyte apoptosis, and >35% of animals died within 7 days of aortic banding. Surviving DN-Raf animals were markedly resistant to the development of cardiac hypertrophy and hypertrophic gene induction in response to transverse aortic constriction. Conclusions—These results establish that Raf-1 kinase activity is essential for cardiac hypertrophy and cardiomyocyte survival in response to pressure overload.

Akt2 Regulates Cardiac Metabolism and Cardiomyocyte Survival

The Akt family of serine-threonine kinases participates in diverse cellular processes, including the promotion of cell survival, glucose metabolism, and cellular protein synthesis. All three known Akt family members, Akt1, Akt2 and Akt3, are expressed in the myocardium, although Akt1 and Akt2 are most abundant. Previous studies demonstrated that Akt1 and Akt3 overexpression results in enhanced myocardial size and function. Yet, little is known about the role of Akt2 in modulating cardiac metabolism, survival, and growth. Here, we utilize murine models with targeted disruption of the akt2 or the akt1 genes to demonstrate that Akt2, but not Akt1, is required for insulin-stimulated 2-[3H]deoxyglucose uptake and metabolism. In contrast, akt2-/- mice displayed normal cardiac growth responses to provocative stimulation, including ligand stimulation of cultured cardiomyocytes, pressure overload by transverse aortic constriction, and myocardial infarction. However, akt2-/- mice were found to be sensitized to cardiomyocyte apoptosis in response to ischemic injury, and apoptosis was significantly increased in the peri-infarct zone of akt2-/- hearts 7 days after occlusion of the left coronary artery. These results implicate Akt2 in the regulation of cardiomyocyte metabolism and survival.

Raf-1 protein kinase is important for progesterone-induced Xenopus oocyte maturation and acts downstream of mos.

In somatic cells, the Raf-1 serine/threonine protein kinase is activated by several polypeptide growth factors. We investigated the role of Raf-1 in progesterone-induced meiotic maturation of Xenopus laevis oocytes. Raf-1 enzymatic activity and phosphorylation (reflected by a mobility shift on sodium dodecyl sulfate gels) were increased in oocytes following progesterone stimulation. The increase in Raf-1 activity was concurrent with an elevation in the activity of mitogen-activated protein (MAP) kinase. When RNA encoding an oncogenic form of Raf-1 (v-Raf) was injected into immature oocytes, MAP kinase mobility shift, germinal vesicle breakdown, and histone H1 phosphorylation increased markedly. When RNA encoding a dominant-negative version of Raf-1 was injected, progesterone-induced oocyte maturation was blocked. When RNA encoding Xenopus mos (mosxe) was injected into oocytes, Raf-1 and MAP kinase mobility shifts were observed after several hours. Also, when antisense mosxe oligonucleotides were injected into oocytes, progesterone-induced Raf-1 and MAP kinase mobility shifts were blocked. Finally, when antisense mosxe oligonucleotides were coinjected with v-Raf RNA into oocytes, histone H1 kinase activation, germinal vesicle breakdown, and MAP kinase mobility shift occurred. These findings suggest that Raf-1 activity is required for progesterone-induced oocyte maturation and that Raf-1 is downstream of mosxe activity.