AKAP12 in astrocytes induces barrier functions in human endothelial cells through protein kinase Czeta

Interactions between astrocytes and blood vessels are essential for the formation and maintenance of the blood-neural barrier (BNB). Astrocyte-derived A-kinase anchor protein 12 (AKAP12) influences BNB formation, but the mechanism of regulation of BNB functions by AKAP12 is not fully understood. We have defined a new pathway of barriergenesis in human retina microvascular endothelial cells (HRMECs) involving astrocytic AKAP12. Treatment of HRMECs with conditioned media from AKAP12-overexpressing astrocytes reduced phosphorylation of protein kinase Czeta (PKCzeta), decreased the levels of vascular endothelial growth factor (VEGF) mRNA and protein, and increased thrombospondin-1 (TSP-1) levels, which led to antiangiogenesis and barriergenesis. Transfection of a small interference RNA targeting PKCzeta decreased VEGF levels and increased TSP-1 levels in HRMECs. Rho is a putative downstream signal of PKCzeta, and inhibition of Rho kinase with a specific inhibitor, Y27632, decreased VEGF levels and increased TSP-1 levels. We therefore suggest that AKAP12 in astrocytes differentially regulates the expression of VEGF and TSP-1 via the inhibition of PKCzeta phosphorylation and Rho kinase activity in HRMECs.     

The roles of phosphatidylinositol 3-kinase and protein kinase Czeta for thrombopoietin-induced mitogen-activated protein kinase activation in primary murine megakaryocytes

Thrombopoietin (TPO) stimulates a network of intracellular signaling pathways that displays extensive cross-talk. We have demonstrated previously that the ERK/mitogen-activated protein kinase pathway is important for TPO-induced endomitosis in primary megakaryocytes (MKs). One known pathway by which TPO induces ERK activation is through the association of Shc with the penultimate phosphotyrosine within the TPO receptor, Mpl. However, several investigators found that the membrane-proximal half of the cytoplasmic domain of Mpl is sufficient to activate ERK in vitro and support base-line megakaryopoiesis in vivo. Using BaF3 cells expressing a truncated Mpl (T69Mpl) as a tool to identify non-Shc/Ras-dependent signaling pathways, we describe here novel mechanisms of TPO-induced ERK activation mediated, in part, by phosphoinositide 3-kinase (PI3K). Similar to cells expressing full-length receptor, PI3K was activated by its incorporation into a complex with IRS2 or Gab2. Furthermore, the MEK-phosphorylating activity of protein kinase Czeta (PKCzeta) was also enhanced after TPO stimulation of T69Mpl, contributing to ERK activity. PKCzeta and PI3K also contribute to TPO-induced ERK activation in MKs, confirming their physiological relevance. Like in BaF3 cells, a TPO-induced signaling complex containing p85PI3K is detectable in MKs expressing T61Mpl and is probably responsible for PI3K activation. These data demonstrate a novel role of PI3K and PKCzeta in steady-state megakaryopoiesis.     

Phosphatidylinositol 3-kinase mediates epidermal growth factor-induced activation of the c-Jun N-terminal kinase signaling pathway.

The signaling events which mediate activation of c-Jun N-terminal kinase (JNK) are not yet well characterized. To broaden our understanding of upstream mediators which link extracellular signals to the JNK pathway, we investigated the role of phosphatidylinositol (PI) 3-kinase in epidermal growth factor (EGF)-mediated JNK activation. In this report we demonstrate that a dominant negative form of PI 3-kinase as well as the inhibitor wortmannin blocks EGF-induced JNK activation dramatically. However, wortmannin does not have an effect on JNK activation induced by UV irradiation or osmotic shock. In addition, a membrane-targeted, constitutively active PI 3-kinase (p110beta) was shown to produce in vivo products and to activate JNK, while a kinase-mutated form of this protein showed no activation. On the basis of these experiments, we propose that PI 3-kinase activity plays a role in EGF-induced JNK activation in these cells.     

Phosphatidylinositol 3-kinase regulates glycosylphosphatidylinositol hydrolysis through PLC-gamma(2) activation in erythropoietin-stimulated cells

Erythropoietin (Epo)-induced glycosylphosphatidylinositol (GPI) hydrolysis was previously described to be correlated with phospholipase C-gamma 2 (PLC-gamma2) activation. Here, we analyzed the involvement of phosphatidylinositol (PtdIns) 3-kinase in GPI hydrolysis through PLC-gamma2 tyrosine phosphorylation in response to Epo in FDC-P1 cells transfected with a wild type (WT) erythropoietin-receptor (Epo-R). We showed that phosphatidylinositol 3-kinase (PtdIns 3-kinase) inhibitor LY294002 inhibits Epo-induced hydrolysis of endogenous GPI and Epo-induced PLC-gamma2 tyrosine phosphorylation in a dose-dependent manner. Wortmannin, another PtdIns 3-kinase inhibitor, also suppressed Epo-induced PLC-gamma2 tyrosine phosphorylation. We also present evidence that PLC-gamma2 translocation to the membrane fraction on Epo stimulation is completely inhibited by LY294002. Upon Epo stimulation, the tyrosine-phosphorylated PLC-gamma2 was found to be associated with the tyrosine-phosphorylated Grb2-associated binder (GAB)2, SHC and SHP2 proteins. LY294002 cell preincubation did not affect GAB2, SHC and SHP2 tyrosine phosphorylation but inhibited the binding of PLC-gamma2 to GAB2 and SHP2. Taken together, these results show that PtdIns 3-kinase controls Epo-induced GPI hydrolysis through PLC-gamma2.     

Phosphatidylinositol 3-kinase activation and interaction with focal adhesion kinase in Escherichia coli K1 invasion of human brain microvascular endothelial cells

Invasion of brain microvascular endothelial cells (BMEC) is a prerequisite for successful crossing of the blood-brain barrier by Escherichia coli K1. We have previously demonstrated the requirement of cytoskeletal rearrangements and activation of focal adhesion kinase (FAK) in E. coli K1 invasion of human BMEC (HBMEC). The current study investigated the role of phosphatidylinositol 3-kinase (PI3K) activation and PI3K interaction with FAK in E. coli invasion of HBMEC. PI3K inhibitor LY294002 blocked E. coli K1 invasion of HBMEC in a dose-dependent manner, whereas an inactive analogue LY303511 had no such effect. In HBMEC, E. coli K1 increased phosphorylation of Akt, a downstream effector of PI3K, which was completely blocked by LY294002. In contrast, non-invasive E. coli failed to activate PI3K. Overexpression of PI3K mutants Deltap85 and catalytically inactive p110 in HBMEC significantly inhibited both PI3K/Akt activation and E. coli K1 invasion of HBMEC. Stimulation of HBMEC with E. coli K1 increased PI3K association with FAK. Furthermore, PI3K/Akt activation was blocked in HBMEC-overexpressing FAK dominant-negative mutants (FRNK and Phe397FAK). These results demonstrated the involvement of PI3K signaling in E. coli K1 invasion of HBMEC and identified a novel role for PI3K interaction with FAK in the pathogenesis of E. coli meningitis.     

Evaluation of the role of protein kinase Czeta in insulin-induced heart 6-phosphofructo-2-kinase activation

A wortmannin-sensitive and insulin-stimulated protein kinase (WISK) that phosphorylates and activates heart 6-phosphofructo-2-kinase (PFK-2) was purified from serum-fed HeLa cells and found to contain protein kinase Czeta (PKCzeta). Both WISK and recombinant PKCzeta were inhibited by a pseudo-substrate peptide inhibitor of PKCzeta. WISK and PKCzeta phosphorylated and activated recombinant heart PFK-2 by increasing its Vmax. The phosphorylation sites in heart PFK-2 for WISK were Ser466 and Thr475, whereas PKCzeta phosphorylated only Thr475. In perfused rat hearts, insulin activated protein kinase B (PKB) 16-fold compared with the untreated controls. However in the same experiments, no change in phosphorylation state of the activation loop Thr410 residue of PKCzeta was observed. By contrast, in incubations of isolated rat epididymal adipocytes, where insulin activated PKB 30-fold compared with the untreated controls, a 50% increase in PKCzeta Thr410 phosphorylation was detected. Lastly in HEK 293T cells transfected with heart PFK-2, co-transfection with a kinase-inactive PKCzeta construct failed to prevent insulin-induced PFK-2 activation. Therefore, it is unlikely that PKCzeta is required for PFK-2 activation by insulin in heart.     

Ca2+ entry via TRPC channels is necessary for thrombin-induced NF-kappaB activation in endothelial cells through AMP-activated protein kinase and protein kinase Cdelta

The transient receptor potential canonical (TRPC) family channels are proposed to be essential for store-operated Ca2+ entry in endothelial cells. Ca2+ signaling is involved in NF-kappaB activation, but the role of store-operated Ca2+ entry is unclear. Here we show that thrombin-induced Ca2+ entry and the resultant AMP-activated protein kinase (AMPK) activation targets the Ca2+-independent protein kinase Cdelta (PKCdelta) to mediate NF-kappaB activation in endothelial cells. We observed that thrombin-induced p65/RelA, AMPK, and PKCdelta activation were markedly reduced by knockdown of the TRPC isoform TRPC1 expressed in human endothelial cells and in endothelial cells obtained from Trpc4 knock-out mice. Inhibition of Ca2+/calmodulin-dependent protein kinase kinase beta downstream of the Ca2+ influx or knockdown of the downstream Ca2+/calmodulin-dependent protein kinase kinase beta target kinase, AMPK, also prevented NF-kappaB activation. Further, we observed that AMPK interacted with PKCdelta and phosphorylated Thr505 in the activation loop of PKCdelta in thrombin-stimulated endothelial cells. Expression of a PKCdelta-T505A mutant suppressed the thrombin-induced but not the TNF-alpha-induced NF-kappaB activation. These findings demonstrate a novel mechanism for TRPC channels to mediate NF-kappaB activation in endothelial cells that involves the convergence of the TRPC-regulated signaling at AMPK and PKCdelta and that may be a target of interference of the inappropriate activation of NF-kappaB associated with thrombosis.     

OxLDL induces mitogen-activated protein kinase activation mediated via PI3-kinase/Akt in vascular smooth muscle cells

Oxidized low-density lipoprotein (OxLDL) is a risk factor in atherosclerosis and stimulates multiple signaling pathways, including activation of phosphatidylinositol 3-kinase (PI3-K)/Akt and p42/p44 mitogen-activated protein kinase (MAPK), which are involved in mitogenesis of vascular smooth muscle cells (VSMCs). We therefore investigated the relationship between PI3-K/Akt and p42/p44 MAPK activation and cell proliferation induced by OxLDL. OxLDL stimulated Akt phosphorylation in a time- and concentration-dependent manner, as determined by Western blot analysis. Phosphorylation of Akt stimulated by OxLDL and epidermal growth factor (EGF) was attenuated by inhibitors of PI3-K (wortmannin and LY294002) and intracellular Ca2+ chelator (BAPTA/AM) plus EDTA. Pretreatment of VSMCs with pertussis toxin, cholera toxin, and forskolin for 24 h also attenuated the OxLDL-stimulated Akt phosphorylation. In addition, pretreatment of VSMCs with wortmannin or LY294002 inhibited OxLDL-stimulated p42/p44 MAPK phosphorylation and [3H]thymidine incorporation. Furthermore, treatment with U0126, an inhibitor of MAPK kinase (MEK)1/2, attenuated the p42/p44 MAPK phosphorylation, but had no effect on Akt activation in response to OxLDL and EGF. Overexpression of p85-DN or Akt-DN mutants attenuated MEK1/2 and p42/p44 MAPK phosphorylation stimulated by OxLDL and EGF. These results suggest that the mitogenic effect of OxLDL is, at least in part, mediated through activation of PI3-K/Akt/MEK/MAPK pathway in VSMCs.     

Phosphatidylinositol 3-kinase is required for integrin-stimulated AKT and Raf-1/mitogen-activated protein kinase pathway activation.

Cell attachment to fibronectin stimulates the integrin-dependent interaction of p85-associated phosphatidylinositol (PI) 3-kinase with integrin-dependent focal adhesion kinase (FAK) as well as activation of the Ras/mitogen-activated protein (MAP) kinase pathway. However, it is not known if this PI 3-kinase-FAK interaction increases the synthesis of the 3-phosphorylated phosphoinositides (3-PPIs) or what role, if any, is played by activated PI 3-kinase in integrin signaling. We demonstrate here the integrin-dependent accumulation of the PI 3-kinase products, PI 3,4-bisphosphate [PI(3,4)P2] and PI(3,4,5)P3, as well as activation of AKT kinase, a serine/threonine kinase that can be stimulated by binding of PI(3,4)P2. The PI 3-kinase inhibitors wortmannin and LY294002 significantly decreased the integrin-induced accumulation of the 3-PPIs and activation of AKT kinase, without having significant effects on the levels of PI(4,5)P2 or tyrosine phosphorylation of paxillin. These inhibitors also reduced cell adhesion/spreading onto fibronectin but had no effect on attachment to polylysine. Interestingly, integrin-mediated Erk-2, Mek-1, and Raf-1 activation, but not Ras-GTP loading, was inhibited at least 80% by wortmannin and LY294002. In support of the pharmacologic results, fibronectin activation of Erk-2 and AKT kinases was completely inhibited by overexpression of a dominant interfering p85 subunit of PI 3-kinase. We conclude that integrin-mediated adhesion to fibronectin results in the accumulation of the PI 3-kinase products PI(3,4)P2 and PI(3,4,5)P3 as well as the PI 3-kinase-dependent activation of the kinases Raf-1, Mek-1, Erk-2, and AKT and that PI 3-kinase may function upstream of Raf-1 but downstream of Ras in integrin activation of Erk-2 MAP and AKT kinases.     

Ethanol sensitizes NF-kappaB activation in pancreatic acinar cells through effects on protein kinase C-epsilon

Although ethanol abuse is the most common cause of pancreatitis, the mechanism of alcohol's effect on the pancreas is not well understood. Previously, we demonstrated that in vitro ethanol treatment of pancreatic acinar cells augmented the CCK-8-induced activation of NF-kappaB, a key signaling system involved in the inflammatory response of pancreatitis. In the present study, we determine the role for individual PKC isoforms in the sensitizing effect of ethanol on NF-kappaB activation. Dispersed rat pancreatic acini were treated with and without ethanol and then stimulated with CCK-8; 100 nM CCK-8 caused both NF-kappaB and PKC-delta, -epsilon, and -zeta activation, whereas 0.1 nM CCK-8 did not increase PKC-epsilon, PKC-zeta, or NF-kappaB activity. CCK-8 (0.1 nM) did activate PKC-delta. PKC-epsilon activator alone did not cause NF-kappaB activation; however, together with 0.1 nM CCK-8, it caused NF-kappaB activation. Ethanol activated PKC-epsilon without affecting other PKC isoforms or NF-kappaB activity. Of note, stimulation of acini with ethanol and 0.1 nM CCK-8 resulted in the activation of PKC-delta, PKC-epsilon, and NF-kappaB. The NF-kappaB activation to 0.1 nM CCK-8 in ethanol-pretreated acini was inhibited by both PKC-delta inhibitor and PKC-epsilon inhibitor. Taken together, these results demonstrate the different modes of activation of PKC isoforms and NF-kappaB in acini stimulated with ethanol, high-dose CCK-8, and low-dose CCK-8, and furthermore suggest that activation of both PKC-epsilon and -delta is required for NF-kappaB activation. These results suggest that ethanol enhances the CCK-8-induced NF-kappaB activation at least in part through its effects on PKC-epsilon.     

Phosphatidylinositol 3-kinase and protein kinase D1 specifically cooperate to negatively regulate the insulin-like growth factor signaling pathway

Insulin receptor substrate-1 (IRS-1) is a key protein in the insulin-like growth factor (IGF) signaling whose tyrosine phosphorylation by the type 1 IGF receptor is necessary for the recruitment and activation of the downstream effectors. Through the analysis of cross-talks occurring between different tyrosine kinase receptor-dependent signaling pathways, we investigated how two growth factors [epidermal growth factor (EGF) and fibroblast growth factor (FGF)] could modulate the IGF-I-induced IRS-1 tyrosine phosphorylation and its downstream signaling. EGF and FGF inhibited IGF-I-stimulated tyrosine phosphorylation of IRS-1 and the subsequent IGF-I-induced phosphatidylinositol 3-kinase (PI 3-kinase) activity. These EGF- and FGF-inhibitory effects were dependent on both PI 3-kinase and protein kinase D1 (PKD1) signaling pathways but independent on the extracellular signal-regulated kinase (ERK) pathway. PKD1, which was activated independently of the PI 3-kinase pathway, associated with IRS-1 in response to EGF or FGF. Unlike PI 3-kinase, PKD1 did not mediate the EGF- or FGF-induced-IRS-1 serine 307 phosphorylation which was described to inhibit IRS-1. Interestingly, specific inhibition of either PI 3-kinase or PKD1 totally impaired EGF- or FGF-induced inhibition of IGF-I-stimulated IRS-1 tyrosine phosphorylation. This indicated that serine 307 phosphorylation of IRS-1 is not sufficient per se to inhibit the IGF signaling pathway and demonstrated for the first time that the negative regulation of IRS-1 requires the coordinated action of PI 3-kinase and PKD1. This further suggests that PKD1 may be an attractive target for innovative strategies that target the IGF signaling pathway.     

Mitochondrial Dok-4 recruits Src kinase and regulates NF-kappaB activation in endothelial cells

The downstream of kinase (Dok) family of adapter proteins consists of at least five members structurally characterized by an NH2-terminal tandem of conserved pleckstrin homology and phosphotyrosine binding domains linked to a unique COOH-terminal region. To determine the role of the novel adapter protein Dok-4 in endothelial cells, we first investigated the cell localization of Dok-4. Most surprisingly, immunofluorescence microscopy, cell fractionation studies, and studies with enhanced green fluorescent protein chimeras showed that wild type Dok-4 (Dok-4-WT) specifically localized in mitochondria. An NH2-terminal deletion mutant of Dok-4 (Dok-4-(deltaN11-29)), which lacks the mitochondrial targeting sequence, could not accumulate in mitochondria. Co-immunoprecipitation revealed an interaction of c-Src with Dok-4-WT in endothelial cells. Most interestingly, overexpression of Dok-4-WT, but not Dok-4-(deltaN1-99), increased mitochondrial c-Src expression, whereas knock-down of endogenous Dok-4 with a small interfering RNA vector greatly inhibited mitochondrial localization of c-Src, suggesting a unique function for Dok-4 as an anchoring protein for c-Src in mitochondria. Dok-4-WT significantly decreased 39-kDa subunit complex I expression. PP2, a specific Src kinase inhibitor, prevented the Dok-4-mediated complex I decrease, suggesting the involvement of Src kinase in regulation of complex I expression. Dok-4-WT enhanced tumor necrosis factor-alpha (TNF-alpha)-mediated reactive oxygen species (ROS) production, supporting the functional relevance of a Dok-4-Src-complex I/ROS signaling pathway in mitochondria. Finally, Dok-4 enhanced TNF-alpha-mediated NF-kappaB activation, whereas this was inhibited by transfection with Dok-4 small interfering RNA. In addition, Dok-4-induced NF-kappaB activation was also inhibited by transfection of a dominant negative form of c-Src. These data suggest a role for mitochondrial Dok-4 as an anchoring molecule for the tyrosine kinase c-Src, and in turn as a regulator of TNF-alpha-mediated ROS production and NF-kappaB activation.     

PI 3-kinase gamma and protein kinase C-zeta mediate RAS-independent activation of MAP kinase by a Gi protein-coupled receptor.

Receptors coupled to the inhibitory G protein Gi, such as that for lysophosphatidic acid (LPA), have been shown to activate MAP kinase through a RAS-dependent pathway. However, LPA (but not insulin) has now been shown to activate MAP kinase in a RAS-independent manner in CHO cells that overexpress a dominant-negative mutant of the guanine nucleotide exchange protein SOS (CHO-DeltaSOS cells). LPA also induced the activation of MAP kinase kinase (MEK), but not that of RAF1, in CHO-DeltaSOS cells. The RAS-independent activation of MAP kinase by LPA was blocked by inhibitors of phosphatidylinositol 3-kinase (PI3K) or by overexpression of a dominant-negative mutant of the gamma isoform of PI3K. Furthermore, LPA induced the activation of the atypical zeta isoform of protein kinase C (PKC-zeta) in CHO-DeltaSOS cells in a manner that was sensitive to wortmannin or to the dominant-negative mutant of PI3Kgamma, and overexpression of a dominant-negative mutant of PKC-zeta inhibited LPA-induced activation of MAP kinase. These observations indicate that Gi protein-coupled receptors induce activation of MEK and MAP kinase through a RAS-independent pathway that involves PI3Kgamma-dependent activation of atypical PKC-zeta.