The Tat protein of human immunodeficiency virus type 1 (HIV-I) trans-activates viral gene expression and is obligatory for virus replication. Tat function is mediated through a sequence termed TAR that comprises part of the 5'-noncoding region of all HIV-1 mRNAs. This region forms a stable stem-loop structure in vitro. Recent evidence indicates that Tat binds directly to the TAR RNA sequence, and this binding is independent of the nucleotide sequence in the loop but dependent on the integrity of the upper stem. We used the electrophoretic mobility-shift assay to identify the sequence and structure specificity of this interaction and its correlation with Tat trans-activation. We show that a 3-nucleotide bulge structure (positions + 23 to + 25) in TAR RNA is important for both Tat interaction with TAR RNA and Tat-mediated trans-activation of gene expression. Single base substitutions at position + 23 that impair Tat-mediated trans-activation in vivo also reduce binding of Tat to TAR in vitro, suggesting that the first uridine residue in the bulge is the critical base for both functions. In contrast, mutations in the loop (positions + 31 to + 34) and the stem (positions + 9 to + 12 and + 49 to + 52), which reduce Tat-mediated trans-activation, had no effect on Tat binding. We also show that a Tat peptide that includes the basic region required for nucleolar localization binds to TAR RNA with the same specificity as the full-length protein. We conclude that Tat binding to TAR is necessary but not sufficient by itself to account for trans-activation.

The differentiation and maturation of skeletal muscle cells into functional fibers is coordinated largely by inductive signals which act through discrete intracellular signal transduction pathways. Recently, the calciumactivated phosphatase calcineurin (PP2B) and the family of transcription factors known as NFAT have been implicated in the regulation of myocyte hypertrophy and fiber type specificity. Here we present an analysis of the intracellular mechanisms which underlie myocyte differentiation and fiber type specificity due to an insulinlike growth factor 1 (IGF-1)-calcineurin-NFAT signal transduction pathway. We demonstrate that calcineurin enzymatic activity is transiently increased during the initiation of myogenic differentiation in cultured C2C12 cells and that this increase is associated with NFATc3 nuclear translocation. Adenovirus-mediated gene transfer of an activated calcineurin protein (AdCnA) potentiates C2C12 and Sol8 myocyte differentiation, while adenovirus-mediated gene transfer of noncompetitive calcineurin-inhibitory peptides (cain or ⌬AKAP79) attenuates differentiation. AdCnA infection was also sufficient to rescue myocyte differentiation in an IGF-depleted myoblast cell line. Using 10T1/2 cells, we demonstrate that MyoD-directed myogenesis is dramatically enhanced by either calcineurin or NFATc3 cotransfection, while a calcineurin inhibitory peptide (cain) blocks differentiation. Enhanced myogenic differentiation directed by calcineurin, but not NFATc3, preferentially specifies slow myosin heavy-chain expression, while enhanced differentiation through mitogen-activated protein kinase kinase 6 (MKK6) promotes fast myosin heavy-chain expression. These data indicate that a signaling pathway involving IGF-calcineurin-NFATc3 enhances myogenic differentiation whereas calcineurin acts through other factors to promote the slow fiber type program.Skeletal muscle cell differentiation is coordinated by endocrine, paracrine, and autocrine inductive factors that activate discrete intracellular signal transduction pathways, resulting in the modulation of transcription factor activity and the reprogramming of gene expression. During embryonic development, the MyoD family of basic helix-loop-helix transcription factors directly regulate myocyte cell specification and differentiation (reviewed in reference 33). The myogenic basic helix-loophelix proteins operate in concert with other transcriptional regulators such as MEF2, serum response factor, and CBP/ p300 to promote myocyte differentiation (17,34,44,49,60). In turn, these transcriptional regulators are themselves regulated by intracellular signaling pathways and phosphorylation cascades.In general, growth factors such as fibroblast growth factor and transforming growth factor ␤ antagonize myocyte differentiation through signaling pathways involving ras, mitogenactivated protein kinase, and protein kinase C (14, 28, 41). Proliferation-inducing transduction pathways enhance AP-1 activity, increase Id expression, and directly attenuate the activity of t...

The Ca 2؉ -calmodulin-activated Ser͞Thr protein phosphatase calcineurin and the downstream transcriptional effectors of calcineurin, nuclear factor of activated T cells, have been implicated in the hypertrophic response of the myocardium. Recently, the calcineurin inhibitory agents cyclosporine A and FK506 have been extensively used to evaluate the importance of this signaling pathway in rodent models of cardiac hypertrophy. However, pharmacologic approaches have rendered equivocal results necessitating more specific or genetic-based inhibitory strategies. In this regard, we have generated Tg mice expressing the calcineurin inhibitory domains of Cain͞Cabin-1 and A-kinase anchoring protein 79 specifically in the heart. ⌬Cain and ⌬A-kinase-anchoring protein Tg mice demonstrated reduced cardiac calcineurin activity and reduced hypertrophy in response to catecholamine infusion or pressure overload. In a second approach, adenoviral-mediated gene transfer of ⌬Cain was performed in the adult rat myocardium to evaluate the effectiveness of an acute intervention and any potential species dependency. ⌬Cain adenoviral gene transfer inhibited cardiac calcineurin activity and reduced hypertrophy in response to pressure overload without reducing aortic pressure. These results provide genetic evidence implicating calcineurin as an important mediator of the cardiac hypertrophic response in vivo.C ardiac hypertrophy is broadly defined as an adaptive enlargement of the myocardium characterized by the growth of individual cardiac myocytes rather than an increase in cell number. Whereas cardiac hypertrophy is a beneficial response that temporarily augments output, sustained hypertrophy often becomes maladaptive and is a leading predictor of future heart failure (1, 2). To understand the molecular mechanisms that underlie adaptive and maladaptive cardiac hypertrophy, investigation has centered around a characterization of the intracellular signal transduction pathways that promote cardiac myocyte growth (3, 4).One such intracellular signaling pathway involves the calciumcalmodulin, Ser͞Thr protein phosphatase calcineurin (PP2B). Sustained elevations in intracellular calcium concentration, in association with calmodulin, directly activate calcineurin phosphatase activity leading to the dephosphorylation and nuclear translocation of a family of transcription factors known as nuclear factor of activated T cells (5, 6).A role for calcineurin and nuclear factor of activated T cells as regulators of cardiac hypertrophy was recently identified (7). Transgenic (Tg) mice expressing an activated calcineurin or a constitutively nuclear nuclear factor of activated T cells c4 factor in the heart demonstrated profound hypertrophy that rapidly progressed to heart failure (7). In vitro, adenoviral-mediated gene transfer of activated calcineurin also promoted hypertrophic growth of neonatal cardiac myocytes (8). Treatment of cultured cardiomyocytes with the calcineurin inhibitory agents cyclosporine A (CsA) or FK506 blocked agonist-induced hypertrop...