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Fluid shear stress generated by blood flow modulates endothelial cell function via specific intracellular signaling events. We showed previously that flow activated the phosphatidylinositol 3-kinase (PI3K), Akt, and endothelial nitric-oxide synthase (eNOS) via Src kinase-dependent teansactivation of vascular endothelial growth factor receptor 2 (VEGFR2). The scaffold protein Gab1 plays an important role in receptor tyrosine kinase-mediated signal transduction. We found here that laminar flow (shear stress = 12 dynes/cm2) rapidly stimulated Gab1 tyrosine phosphorylation in both bovine aortic endothelial cells and human umbilical vein endothelial cells, which correlated with activation of Akt and eNOS. Gab1 phosphorylation as well as activation of Akt and eNOS by flow was inhibited by the Src kinase inhibitor PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) and VEGFR2 kinase inhibitors SU1498 and VTI, suggesting that flow-mediated Gab1 phosphorylation is Src kinase-dependent and VEGFR2-dependent. Tyrosine phosphorylation of Gab1 by flow was functionally important, because flow stimulated the association of Gab1 with the PI3K subunit p85 in a time-dependent manner. Furthermore, transfection of a Gab1 mutant lacking p85 binding sites inhibited flow-induced activation of Akt and eNOS. Finally, knockdown of endogenous Gab1 by small interference RNA abrogated flow activation of Akt and eNOS. These data demonstrate a critical role of Gab1 in flow-stimulated PI3K/Akt/eNOS signal pathway in endothelial cells. [ABSTRACT FROM AUTHOR]

Growth factors and their downstream receptor tyrosine kinases (RTKs) mediate a number of biological processes controlling cell function. Adaptor (docking) proteins, which consist exclusively of domains and motifs that mediate molecular interactions, link receptor activation to downstream effectors. Recent studies have revealed that Grb2-associated-binders (Gab) family members (including Gab1, Gab2, and Gab3), when phosphorylated on tyrosine residues, provide binding sites for multiple effector proteins, such as Src homology-2 (SH2)-containing protein tyrosine phosphatase 2 (SHP2) and phosphatidylinositol 3-kinase (PI3K) regulatory subunit p85, thereby playing important roles in transducing RTKs-mediated signals into pathways with diversified biological functions. Here, we provide an up-to-date overview on the domain structure and biological functions of Gab1, the most intensively studied Gab family protein, in growth factor signaling and biological functions, with a special focus on angiogenesis. [ABSTRACT FROM AUTHOR]

Dynamic nucleocytoplasmic shuttling of class IIa histone deacetylases (HDACs) is a fundamental mechanism regulating gene transcription. Recent studies have identified several protein kinases that phosphorylate HDAC5, leading to its exportation from the nucleus. However, the negative regulatory mechanisms for HDAC5 nuclear exclusion remain largely unknown. Here we show that cAMP-activated protein kinase A (PKA) specifically phosphorylates HDAC5 and prevents its export from the nucleus, leading to suppression of gene transcription. PKA interacts directly with HDAC5 and phosphorylates HDAC5 at serine 280, an evolutionarily conserved site. Phosphorylation of HDAC5 by PKA interrupts the association of HDAC5 with protein chaperone 14-3-3 and hence inhibits stress signal-induced nuclear export of HDAC5. An HDAC5 mutant that mimics PKA-dependent phosphorylation localizes in the nucleus and acts as a dominant inhibitor for myocyte enhancer factor 2 transcriptional activity. Molecular manipulations of HDAC5 show that PKA-phosphorylated HDAC5 inhibits cardiac fetal gene expression and cardiomyocyte hypertrophy. Our findings identify HDAC5 as a substrate of PKA and reveal a cAMP/PKA-dependent pathway that controls HDAC5 nucleocytoplasmic shuttling and represses gene transcription. This pathway may represent a mechanism by which cAMP/PKA signaling modulates a wide range of biological functions and human diseases such as cardiomyopathy. [ABSTRACT FROM AUTHOR]

Vascular endothelial growth factor (VEGF) is essential for normal and pathological angiogenesis. However, the signaling pathways linked to gene regulation in VEG F-induced angiogenesis are not fully understood. Here we demonstrate a critical role of protein kinase D (PKD) and histone deacetylase 5 (HDAC5) in VEGF-induced gene expression and angiogenesis. We found that VEGF stimulated HDAC5 phosphorylation and nuclear export in endothelial cells through a VEGF receptor 2-phospholipase Cγ-protein kinase C-PKD-dependent pathway. We further showed that the PKD-HDAC5 pathway mediated myocyte enhancer factor-2 transcriptional activation and a specific subset of gene expression in response to VEGF, including NR4A1, an orphan nuclear receptor involved in angiogenesis. Specifically, inhibition of PKD by overexpression of the PKD kinase-negative mutant prevents VEGF-induced HDAC5 phosphorylation and nuclear export as well as NR4A1 induction. Moreover, a mutant of HDAC5 specifically deficient in PKD-dependent phosphorylation inhibited VEGF-mediated NR4A1 expression, endothelial cell migration, and in vitro angiogenesis. These findings suggest that the PKD-HDAC5 pathway plays an important role in VEGF regulation of gene transcription and angiogenesis. [ABSTRACT FROM AUTHOR]

Blood flow modulates endothelial cell (EC) functions through specific signaling events. Previous data show that flow stimulates SHP2 translocation to cell membranes and binding to phosphotyrosine proteins. Flow-induced ERK½ phosphorylation depends on SHP2 phosphatase activity and SHP2 binding to phospho-PE-CAM1 (platelet endothelial adhesion molecule 1), suggesting that SHP2 forms a signaling module with PE-CAM1. We hypothesized that flow induces assembly of the multi-protein complexes with SHP2 that are required for downstream signaling. ECs were exposed to flow for 10 min, and endogenous SHP2 was immunoprecipitated. SHP2-associated proteins were analyzed by SDS-PAGE and identified by mass spectrometry. Tie2 and several known SHP2-binding proteins were identified in flow-induced SHP2 complexes. Flow significantly increased tyrosine phosphorylation of both Tie2 and PE-CAM1 and their association with SHP2. To evaluate their functional roles, ECs were treated with Tie2 or PECAM1 small interfering RNA (siRNA). Tie2 and PE-CAM1 expression decreased >80% after siRNA treatment, and flow-stimulated phosphorylation of ERK½, Akt, and endothelial nitric oxide synthase was significantly inhibited by Tie2 and PECAM1 siRNA. Tie2 phosphorylation by flow was significantly inhibited by PE-CAM1 siRNA treatment. These results establish Tie2 transactivation via PECAM1 as an early event in flow-mediated mechanotransduction and suggest an important role for a PECAM1-SHP2-Tie2 pathway in flow-mediated signal transduction. [ABSTRACT FROM AUTHOR]

Angiotensin II (Ang II) induces the phenotypic modulation and hypertrophy of vascular smooth muscle cells (VSMCs), which is implicated in the pathogenesis of hypertension and atherosclerosis. Here we established a novel role of histone deacetylases 5 (HDAC5) in Ang II-induced VSMC hypertrophy. We showed that Ang II rapidly stimulated phosphorylation of HDAC5 (p-HDAC5) on Ser259/498 sites in a time- and dose- dependent manner in rat aortic VSMCs. Ang II receptor 1 mediated p-HDAC5. Protein kinase C (PKC) inhibitors, GF109203X and Gö6983, inhibited p-HDAC5. Using pharmacological inhibitor, siRNA and adenovirus encoding kinase-negative mutant of protein kinase D (PKD) demonstrated that PKD was essential for p-HDAC5 by Ang II. Moreover, we showed that Ang II stimulated HDAC5 nuclear export, which was dependent on its phosphorylation via PKC/PKD pathway, because HDAC5 nuclear export was abolished by inhibiting PKC and PKD and by infection of adenovirus encoding HDAC5 S259/498A mutant. Furthermore, both PKD inhibition and HDAC5 S259/498A mutant blocked Ang II-induced myocyte enhancer factor 2 (MEF2) transcriptional activation. Importantly, Ang II promoted VSMC protein synthesis in PKD and HDAC5-dependent manner. Our findings reveal for the first time that PKD and HDAC5 play a key role in Ang II-induced VSMC hypertrophy, and suggest that PKD and HDAC5 may emerge as potential targets for cardiovascular diseases. [ABSTRACT FROM AUTHOR]

Vascular endothelial growth factor (VEGF) is essential for many angiogenic processes. Histone deacetylases (HDACs) have emerged as key transcriptional regulators. Here we showed that VEGF induced Protein kinase D (PKD)-dependent HDAC5 phosphorylation and nuclear export in endothelial cells. VEGF rapidly, stimulated HDAC5 phosphorylation (p-HDAC5) at Ser259/498 residues in a time- and dose-dependent manner in bovine aortic endothelial cells and human umbilical vein endothelial cells. VEGF-induced p-HDAC5 was mediated via VEGF receptor 2, phospholipase Cγ (PLCγ), protein kinase C (PKC) and PKD pathway. Specifically, PLCγ and PKCs inhibitors diminished p-HDAC5. Inhibiting PKD by siRNA and adenovirus encoding kinase-negative mutant blocked p-HDACS. Furthermore, VEGF stimulated nuclear export of HDAC5 in a phosphorylation-dependent manner, which leads to myocyte enhance factors-2 (MEF2) transcriptional activation. Active PKD stimulated HDAC5 nuclear export and MEF2 activation. In contrast, inhibiting PKD prevented VEGF-mediated HDAC5 nuclear export and MEF2 activation. Moreover, adenovirus encoding HDAC5 S258/498A mutant abolished VEGF-mediated HDAC5 nuclear export and MEF2 activation. Our findings reveal a novel role of PKD and HDAC5 in VEGF regulation of gene transcription and suggest that PKD and HDAC5 may emerge as potential therapeutic targets for pathological angiogenesis. [ABSTRACT FROM AUTHOR]