Protein kinase D (PKD), also called protein kinase C (PKC)μ, is a serine-threonine kinase that is involved in diverse areas of cellular function such as lymphocyte signaling, oxidative stress, and protein secretion. After identifying a putative PKD phosphorylation site in the Toll/IL-1R domain of TLR5, we explored the role of this kinase in the interaction between human TLR5 and enteroaggregative Escherichia coli flagellin in human epithelial cell lines. We report several lines of evidence that implicate PKD in TLR5 signaling. First, PKD phosphorylated the TLR5-derived target peptide in vitro, and phosphorylation of the putative target serine 805 in HEK 293T cell-derived TLR5 was identified by mass spectrometry. Furthermore, mutation of serine 805 to alanine abrogated responses of transfected HEK 293T cells to flagellin. Second, TLR5 interacted with PKD in coimmunoprecipitation experiments, and this association was rapidly enhanced by flagellin treatment. Third, pharmacologic inhibition of PKC or PKD with Gö6976 resulted in reduced expression and secretion of IL-8 and prevented the flagellin-induced activation of p38 MAPK, but treatment with the PKC inhibitor Gö6983 had no significant effects on these phenotypes. Finally, involvement of PKD in the p38-mediated IL-8 response to flagellin was confirmed by small hairpin RNA-mediated gene silencing. Together, these results suggest that phosphorylation of TLR5 by PKD may be one of the proximal elements in the cellular response to flagellin, and that this event contributes to p38 MAPK activation and production of inflammatory cytokines in epithelial cells.

Abstract-The diabetic heart switches to exclusively using fatty acid (FA) for energy supply and does so by multiple mechanisms including hydrolysis of lipoproteins by lipoprotein lipase (LPL) positioned at the vascular lumen. We determined the mechanism that leads to an increase in LPL after diabetes. Diazoxide (DZ), an agent that decreases insulin secretion and causes hyperglycemia, induced a substantial increase in LPL activity at the vascular lumen. This increase in LPL paralleled a robust phosphorylation of Hsp25, decreasing its association with PKC␦, allowing this protein kinase to phosphorylate and activate protein kinase D (PKD), an important kinase that regulates fission of vesicles from the golgi membrane. Rottlerin, a PKC␦ inhibitor, prevented PKD phosphorylation and the subsequent increase in LPL. Incubating control myocytes with high glucose and palmitic acid (GluϩPA) also increased the phosphorylation of Hsp25, PKC␦, and PKD in a pattern similar to that seen with diabetes, in addition to augmenting LPL activity. In myocytes in which PKD was silenced or a mutant form of PKC␦ was expressed, high GluϩPA were incapable of increasing LPL. Moreover, silencing of cardiomyocyte Hsp25 allowed phorbol 12-myristate 13-acetate to elicit a significant phosphorylation of PKC␦, an appreciable association between PKC␦ and PKD, and a vigorous activation of PKD. As these cells also demonstrated an additional increase in LPL, our data imply that after diabetes, PKD control of LPL requires dissociation of Hsp25 from PKC␦, association between PKC␦ and PKD, and vesicle fission. Results from this study could help in restricting cardiac LPL translocation, leading to strategies that overcome contractile dysfunction after diabetes. Key Words: heat shock protein Ⅲ protein kinase C Ⅲ hyperglycemia Ⅲ hyperlipidemia Ⅲ vesicles C ardiac muscle has a high demand for energy and uses multiple substrates, including fatty acid (FA), carbohydrate, amino acids, and ketones. 1 Among these substrates, carbohydrate and FA are the major sources from which the heart derives most of its energy. In a normal heart, whereas glucose and lactate account for approximately 30% of energy provided to the cardiac muscle, 70% of ATP generation is through FA oxidation. 2 FA delivery and utilization by the heart involves: (1) release from adipose tissue and transport to the heart after complexing with albumin, 3 (2) provision through breakdown of endogenous cardiac triglyceride (TG) stores, 4 (3) internalization of whole lipoproteins, 5 and (4) hydrolysis of circulating TG-rich lipoproteins to FA by lipoprotein lipase (LPL) positioned at the endothelial surface of the coronary lumen. 6 The molar concentration of FA bound to albumin is Ϸ10-fold less than that of FA in lipoprotein-TG, 7 and LPL-mediated hydrolysis of circulating TG-rich lipoproteins to FA is suggested to be the principal source of FA for cardiac utilization. 8 Coronary endothelial cells do not synthesize LPL. 9 In the heart, this enzyme is produced in cardiomyocytes and subsequently se...

Glycogen is an immediate source of glucose for cardiac tissue to maintain its metabolic homeostasis. However, its excess brings about cardiac structural and physiological impairments. Previously, we have demonstrated that in hearts from dexamethasone (Dex)-treated animals, glycogen accumulation was enhanced. We examined the influence of 5′-AMP-activated protein kinase (AMPK) on glucose entry and glycogen synthase as a means of regulating the accumulation of this stored polysaccharide. After Dex, cardiac tissue had a limited contribution toward the development of whole body insulin resistance. Measurement of glucose transporter 4 (GLUT4) at the plasma membrane revealed an excess presence of this transporter protein at this location. Interestingly, this was accompanied by an increase in GLUT4 in the intracellular membrane fraction, an effect that was well correlated with increased GLUT4 mRNA. Both total and phosphorylated AMPK increased after Dex. Immunoprecipitation of Akt substrate of 160 kDa (AS160) followed by Western blot analysis demonstrated no change in Akt phosphorylation at Ser473 and Thr308 in Dex-treated hearts. However, there was a significant increase in AMPK phosphorylation at Thr172, which correlated well with AS160 phosphorylation. In Dex-treated hearts, there was a considerable reduction in the phosphorylation of glycogen synthase, whereas glycogen synthase kinase-3-β phosphorylation was augmented. Our data suggest that AMPK-mediated glucose entry combined with the activation of glycogen synthase and a reduction in glucose oxidation (Qi et al., Diabetes 53: 1790–1797, 2004) act together to promote glycogen storage. Should these effects persist chronically in the heart, they may explain the increased morbidity and mortality observed with long-term excesses in endogenous or exogenous glucocorticoids.

Wnt signaling is mediated by three classes of receptors, Frizzled, Ryk, and Ror. In Caenorhabditis elegans, Wnt signaling regulates the anterior/posterior polarity of the P7.p vulval lineage, and mutations in lin-17/Frizzled cause loss or reversal of P7.p lineage polarity. We found that pak-1/Pak (p21-activated kinase), along with putative activators of Pak, nck-1/Nck, and ced-10/Rac, regulates P7.p polarity. Mutations in these genes suppress the polarity defect of lin-17 mutants. Furthermore, mutations in pak-1, nck-1, and ced-10 cause constitutive dauer formation at 27°C, a phenotype also observed in egl-20/Wnt and cam-1/Ror mutants. In HEK293T cells, Pak1 can antagonize canonical Wnt signaling. Moreover, overexpression of Ror2 leads to phosphorylation of Pak1. Together, these results indicate that Pak interacts with Wnt signaling to regulate tissue polarity and gene expression.cell polarity | β-catenin | T-cell factor | patterning | asymmetry

(LPL), an enzyme that hydrolyzes lipoproteins to FA. We examined the mechanisms by which DEX augments cardiac LPL. DEX was injected in rats, and hearts were removed, or isolated cardiomyocytes were incubated with DEX (0 -8 h), for measurement of LPL activity and Western blotting. Acute DEX induced whole body insulin resistance, likely an outcome of a decrease in insulin signaling in skeletal muscle, but not cardiac tissue. The increase in luminal LPL activity after DEX was preceded by rapid nongenomic alterations, which included phosphorylation of AMPK and p38 MAPK, that led to phosphorylation of heat shock protein (HSP)25 and actin cytoskeleton rearrangement, facilitating LPL translocation to the myocyte cell surface. Unlike its effects in vivo, although DEX activated AMPK and p38 MAPK in cardiomyocytes, there was no phosphorylation of HSP25, nor was there any evidence of F-actin polymerization or an augmentation of LPL activity up to 8 h after DEX. Combining DEX with insulin appreciably enhanced cardiomyocyte LPL activity, which closely mirrored a robust elevation in phosphorylation of HSP25 and F-actin polymerization. Silencing of p38 MAPK, inhibition of PI 3-kinase, or preincubation with cytochalasin D prevented the increases in LPL activity. Our data suggest that, following DEX, it is a novel, rapid, nongenomic phosphorylation of stress kinases that, together with insulin, facilitates LPL translocation to the myocyte cell surface.