No abstract

Insulin stimulates the activity of mitogen-activated protein kinase (MAPK) via its upstream activator, MAPK kinase (MEK), a dual specificity kinase that phosphorylates MAPK on threonine and tyrosine. The potential role of MAPK activation in insulin action was investigated with the specific MEK inhibitor PD98059. Insulin stimulation of MAPK activity in 3T3-L1 adipocytes (2.7-fold) and L6 myotubes (1.4-fold) was completely abolished by pretreatment of cells with the MEK inhibitor, as was the phosphorylation of MAPK and pp90Rsk, and the transcriptional activation of c-fos. Insulin receptor autophosphorylation on tyrosine residues and activation of phosphatidylinositol 3'-kinase were unaffected. Pretreatment of cells with PD98059 had no effect on basal and insulin-stimulated glucose uptake, lipogenesis, and glycogen synthesis. Glycogen synthase activity in extracts from 3T3-L1 adipocytes and L6 myotubes was increased 3-fold and 1.7-fold, respectively, by insulin. Pretreatment with 10 microM PD98059 was without effect. Similarly, the 2-fold activation of protein phosphatase 1 by insulin was insensitive to PD98059. These results indicate that stimulation of the MAPK pathway by insulin is not required for many of the metabolic activities of the hormone in cultured fat and muscle cells.

The zinc finger-containing transcription factor GATA4 has been implicated as a critical regulator of multiple cardiac-expressed genes as well as a regulator of inducible gene expression in response to hypertrophic stimulation. Here we demonstrate that GATA4 is itself regulated by the mitogen-activated protein kinase signaling cascade through direct phosphorylation. Site-directed mutagenesis and phospho-specific GATA4 antiserum revealed serine 105 as the primary site involved in agonist-induced phosphorylation of GATA4. Infection of cultured cardiomyocytes with an activated MEK1-expressing adenovirus induced robust phosphorylation of serine 105 in GATA4, while a dominant-negative MEK1-expressing adenovirus blocked agonistinduced phosphorylation of serine 105, implicating extracellular signal-regulated kinase (ERK) as a GATA4 kinase. Indeed, bacterially purified ERK2 protein directly phosphorylated purified GATA4 at serine 105 in vitro. Phosphorylation of serine 105 enhanced the transcriptional potency of GATA4, which was sensitive to U0126 (MEK1 inhibitor) but not SB202190 (p38 inhibitor). Phosphorylation of serine 105 also modestly enhanced the DNA binding activity of bacterially purified GATA4. Finally, induction of cardiomyocyte hypertrophy with an activated MEK1-expressing adenovirus was blocked with a dominant-negative GATA4-engrailed-expressing adenovirus. These results suggest a molecular pathway whereby MEK1-ERK1/2 signaling regulates cardiomyocyte hypertrophic growth through the transcription factor GATA4 by direct phosphorylation of serine 105, which enhances DNA binding and transcriptional activation.

Specification and differentiation of the cardiac muscle lineage appear to require a combinatorial network of many factors. The cardiac muscle-restricted homeobox protein Csx/Nkx2.5 (Csx) is expressed in the precardiac mesoderm as well as the embryonic and adult heart. Targeted disruption of Csx causes embryonic lethality due to abnormal heart morphogenesis. The zinc finger transcription factor GATA4 is also expressed in the heart and has been shown to be essential for heart tube formation. GATA4 is known to activate many cardiac tissue-restricted genes. In this study, we tested whether Csx and GATA4 physically associate and cooperatively activate transcription of a target gene. Coimmunoprecipitation experiments demonstrate that Csx and GATA4 associate intracellularly. Interestingly, in vitro protein-protein interaction studies indicate that helix III of the homeodomain of Csx is required to interact with GATA4 and that the carboxy-terminal zinc finger of GATA4 is necessary to associate with Csx. Both regions are known to directly contact the cognate DNA sequences. The promoter-enhancer region of the atrial natriuretic factor (ANF) contains several putative Csx binding sites and consensus GATA4 binding sites. Transient-transfection assays indicate that Csx can activate ANF reporter gene expression to the same extent that GATA4 does in a DNA binding site-dependent manner. Coexpression of Csx and GATA4 synergistically activates ANF reporter gene expression. Mutational analyses suggest that this synergy requires both factors to fully retain their transcriptional activities, including the cofactor binding activity. These results demonstrate the first example of homeoprotein and zinc finger protein interaction in vertebrates to cooperatively regulate target gene expression. Such synergistic interaction among tissue-restricted transcription factors may be an important mechanism to reinforce tissue-specific developmental pathways.Increasing evidence suggests that multiple trans-acting factors and cis-acting elements cooperatively regulate the expression of cardiac muscle-specific genes (reviewed in references 28 and 36), unlike skeletal muscle myogenesis where myogenic basic helix-loop-helix factors can activate the entire myogenic program (reviewed by Olson and Klein [37a]). For example, the cardiac ␣-myosin heavy chain gene (␣-MHC) is synergistically activated by myocyte-specific enhancer factor 2 (MEF2) and thyroid hormone receptor, and this activation depends on the binding of each factor to the DNA target sequences (27). Multiple transcription factors, such as E-box and CArG-box binding factors and Sp1, are required for the muscle-specific expression of the cardiac ␣-actin gene (37b). Cardiac myosin light chain 2v (MLC2v) gene expression appears to depend on several factors, including YB-1 and CARP (44,45).Homeobox genes have been studied extensively in many animal species, where they play fundamental roles in specifying cell fate and positional identity in embryos. The nk-4/msh-2 Drosophila gene, tinman, has been ...

The precise mechanism by which insulin regulates glucose metabolism is not fully understood. However, it is known that insulin activates two enzymes, phosphatidylinositol 3'-kinase (PI 3'-K) and mitogen-activated protein kinase (MAPK), which may be involved in stimulating the metabolic effects of insulin. The role of these enzymes in glucose metabolism was examined by comparing the effects of insulin, platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) in 3T3-L1 adipocytes. Treatment of the cells with PDGF or EGF for 5 min increased the MAPK activity 3-5-fold, while insulin treatment produced a 2.5-fold increase. The MAPK activity remained elevated for 1 h after either PDGF or insulin treatment. PDGF and insulin, but not EGF, caused a transient increase in the amount PI 3'-K activity coprecipitated with tyrosine phosphorylated proteins. Although PDGF and insulin caused a similar increase in the activities of these two enzymes, only insulin caused substantial increases in glucose utilization. Insulin increased the transport of glucose and the synthesis of lipid 4- and 17-fold, respectively, while PDGF did not affect these processes significantly. Glycogen synthesis was increased 15-fold in response to insulin and only 3-fold in response to PDGF. Thus, the activation of MAPK and PI 3'-K are not sufficient for the complete stimulation of glucose transport, lipid synthesis, or glycogen synthesis by hormones in 3T3-L1 adipocytes, suggesting a requirement for other signaling mechanisms that may be uniquely responsive to insulin.