Mitogen-activated Protein Kinase Phosphatidylinositol 3-kinase Research Articles

PGC-1α Serine 570 Phosphorylation and GCN5-mediated Acetylation by Angiotensin II Drive Catalase Down-regulation and Vascular Hypertrophy

Angiotensin II (Ang II) is a pleuripotential hormone that is important in the pathophysiology of multiple conditions including aging, cardiovascular and renal diseases, and insulin resistance. Reactive oxygen species (ROS) are important mediators of Ang II-induced signaling generally and have a well defined role in vascular hypertrophy, which is inhibited by overexpression of catalase, inferring a specific role of H(2)O(2). The molecular mechanisms are understood incompletely. The transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1 alpha) is a key regulator of energy metabolism and ROS-scavenging enzymes including catalase. We show that Ang II stimulates Akt-dependent PGC-1 alpha serine 570 phosphorylation, which is required for the binding of the histone acetyltransferase GCN5 (general control nonderepressible 5) to PGC-1 alpha and for its lysine acetylation. These sequential post-translational modifications suppress PGC-1 alpha activity and prevent its binding to the catalase promoter through the forkhead box O1 transcription factor, thus decreasing catalase expression. We demonstrate that overexpression of the phosphorylation-defective mutant PGC-1 alpha (S570A) prevents Ang II-induced increases in H(2)O(2) levels and hypertrophy ([(3)H]leucine incorporation). Knockdown of PGC-1 alpha by small interfering RNA promotes basal and Ang II-stimulated ROS and hypertrophy, which is reversed by polyethylene glycol-conjugated catalase. Thus, endogenous PGC-1 alpha is a negative regulator of vascular hypertrophy by up-regulating catalase expression and thus reducing ROS levels. We provide novel mechanistic insights by which Ang II may mediate its ROS-dependent pathophysiologic effects on multiple cardiometabolic diseases.

ADP-ribosylation Factor 1 Controls the Activation of the Phosphatidylinositol 3-Kinase Pathway to Regulate Epidermal Growth Factor-dependent Growth and Migration of Breast Cancer Cells

Activation of intracellular signaling pathways by growth factors is one of the major causes of cancer development and progression. Recent studies have demonstrated that monomeric G proteins of the Ras family are key regulators of cell proliferation, migration, and invasion. Using an invasive breast cancer cell lines, we demonstrate that the ADP-ribosylation factor 1 (ARF1), a small GTPase classically associated with the Golgi, is an important regulator of the biological effects induced by epidermal growth factor. Here, we show that this ARF isoform is activated following epidermal growth factor stimulation and that, in MDA-MB-231 cells, ARF1 is found in dynamic plasma membrane ruffles. Inhibition of endogenous ARF1 expression results in the inhibition of breast cancer cell migration and proliferation. The underlying mechanism involves the activation of the phosphatidylinositol 3-kinase pathway. Our data demonstrate that depletion of ARF1 markedly impairs the recruitment of the phosphatidylinositol 3-kinase catalytic subunit (p110alpha) to the plasma membrane, and the association of the regulatory subunit (p85alpha) to the activated receptor. These results uncover a novel molecular mechanism by which ARF1 regulates breast cancer cell growth and invasion during cancer progression.

Cell-specific Regulation of Androgen Receptor Phosphorylation in Vivo

The biological ramifications of phosphorylation of the androgen receptor (AR) are largely unknown. To examine the phosphorylation of AR at serine 213, a putative substrate for Akt, a phosphorylation site-specific antibody was generated. The use of this antibody indicated that AR Ser-213 is phosphorylated in vivo and that phosphorylation is tightly regulated in a cell type-specific manner. Furthermore, Ser-213 phosphorylation took place with rapid kinetics and was inhibited by the phosphatidylinositol 3-kinase inhibitor LY294002. Phosphorylation occurred in response to R1881 and dihydrotestosterone but weakly if at all in response to testosterone. It did not occur in response to AR antagonists or growth factor stimulation in the absence of an AR agonist. Transcription assays using an AR-responsive reporter gene construct showed that activated phosphatidylinositol 3-kinase inhibited transcription mediated by wild type AR but not that of a mutant AR variant (S213A), which could not be phosphorylated at Ser-213. By immunohistochemistry, the AR Ser(P)-213 antigen was detected in prostate epithelial but not stromal cells despite the fact that an antibody recognizing both phosphorylated and non-phosphorylated forms of AR demonstrates that AR is present in both cell types as expected. In fetal tissue the AR-Ser(P)-213 antigen was present in epithelial cells of the urogenital sinus when endogenous androgen levels were high and activated Akt was prevalent, but absent at a later stage of development when endogenous androgen levels were low and Akt activation was minimal. Immunoreactivity was evident in differentiated cells lining the lumen of the urogenital sinus but not in rapidly dividing, Ki67 positive cells within the developing prostate or stromal tissue, suggesting that site-specific phosphorylation of AR Ser-213 by cellular kinases occurs in a non-proliferating cellular milieu.

Insulin-like Growth Factor-I Induces Renal Cell Hypertrophy via a Calcineurin-dependent Mechanism

Insulin-like growth factor-I (IGF-I) may play an important role in the development of renal hypertrophy. In this study we determined the effect of IGF-I on cultured mesangial cells (MCs) and examined activation of key signaling pathways. IGF-I induced hypertrophy as determined by an increase in cell size and an increase in protein to DNA ratio and increased accumulation of extracellular matrix (ECM) proteins. IGF-I also activated both Erk1/Erk2 MAPK and phosphatidylinositol 3-kinase (PI3K) in MCs. Inhibition of either MAPK or PI3K, however, had no effect on IGF-I-induced hypertrophy or ECM production. Next, we examined the effect of IGF-I on activation of the calcium-dependent phosphatase calcineurin. IGF-I treatment stimulated calcineurin activity and increased the protein levels of calcineurin and the calcineurin binding protein, calmodulin. Cyclosporin A, an inhibitor of calcineurin, blocked both IGF-I-mediated hypertrophy and up-regulation of ECM. In addition, calcineurin resulted in sustained Akt activation, indicating possible cross-talk with other signaling pathways. Finally, IGF-I treatment resulted in the calcineurindependent nuclear localization of NFATc1. Therefore, IGF-I induces hypertrophy and increases ECM accumulation in MCs. IGF-I-mediated hypertrophy is associated with activation of Erk1/Erk2 MAPK and PI3K but does not require either of these pathways. Instead, IGF-I mediates hypertrophy via a calcineurin-dependent pathway.