Bryon D. Grove

Intracellular Distribution of Gravin, a PKA and PKC Binding Protein, in Vascular Endothelial Cells

Gravin, a high-molecular-weight protein expressed widely in tissues and cells, is upregulated in cultured endothelial cells under conditions which suggest that it may play a role in wound repair and vascular development. In the current study, we examined the intracellular distribution of gravin to determine if it is associated with the cytoskeleton or with another intracellular compartment. Immunofluorescence microscopy of human umbilical vein endothelial cells (HUVEC) revealed that gravin had a punctate staining distribution that extended to the cell margin and did not appear to colocalize with stress fibers, microtubules, and intermediate filaments. Moreover, disruption of the cytoskeletal structures with either cytochalasin D or colchicine did not alter gravin distribution. However, confocal and immunoelectron microscopy clearly revealed that gravin was concentrated at the cell margin in close association with the plasma membrane. Immunoprecipitation of gravin from endothelial cell lysates resulted in coprecipitation of protein kinase activity that could be eluted from the immunoprecipitates with cAMP and that was inhibitable with a PKA-specific inhibitor. An anti-PKA catalytic subunit antibody reacted with a 40-kD band on immunoblots of the cAMP eluate. Immunoblots of the immunoprecipitates further revealed that PKCα coprecipitated with gravin from endothelial cell lysates. This study indicates that gravin is associated with either the plasma membrane or the membrane skeleton and may play a role in endothelial wound healing by targeting PKA and PKC to specific membrane-associated sites and regulating PKA/PKC-dependent cellular activities associated with endothelial wound healing.

Gravin dynamics regulates the subcellular distribution of PKA.

Gravin, a multivalent A-kinase anchoring protein (AKAP), localizes to the cell periphery in several cell types and is postulated to target PKA and other binding partners to the plasma membrane. An N-terminal myristoylation sequence and three regions rich in basic amino acids are proposed to mediate this localization. Reports indicating that phorbol ester affects the distribution of SSeCKS, the rat orthologue of gravin, further suggest that PKC may also regulate the subcellular distribution of gravin, which in turn may affect PKA distribution. In this study, quantitative confocal microscopy of cells expressing full-length and mutant gravin-EGFP constructs lacking the proposed targeting domains revealed that either the N-myristoylation site or the polybasic regions were sufficient to target gravin to the cell periphery. Moreover, phorbol ester treatment induced redistribution of gravin-EGFP from the cell periphery to a juxtanuclear vesicular compartment, but this required the presence of the N-myristoylation site. Confocal microscopy further revealed that not only did gravin-EGFP target a PKA RII-ECFP construct to the cell periphery, but PKC activation resulted in redistribution of the gravin and PKA constructs to the same subcellular site. It is postulated that this dynamic response by gravin to PKC activity may mediate PKC dependent control of PKA activity.

The 27-kDa Heat Shock Protein Confers Cytoprotective Effects through a β2-Adrenergic Receptor Agonist-Initiated Complex with β-Arrestin

Heat shock proteins represent an emerging model for the coordinated, multistep regulation of apoptotic signaling events. Although certain aspects of the biochemistry associated with heat shock protein cytoprotective effects are known, little information is found describing the regulation of heat shock protein responses to harmful stimuli. During screening for noncanonical β adrenergic receptor signaling pathways in human urothelial cells, using mass spectroscopy techniques, an agonist-dependent interaction with β-arrestin and the 27-kDa heat shock protein was observed in vitro. Formation of this β-arrestin/Hsp27 complex in response to the selective β adrenergic receptor agonist isoproterenol, was subsequently confirmed in situ by immunofluorescent colocalization studies. Radioligand binding techniques characterized a homogeneous population of the β2 adrenergic receptor subtype expressed on these cells. Using terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling, immunoblot analysis and quantitation of caspase-3 activity to detect apoptosis, preincubation of these cells with isoproterenol was found to be sufficient for protection against programmed cell death initiated by staurosporine. RNA interference strategies confirmed the necessity for Hsp27 as well as both β-arrestin isoforms to confer this cytoprotective consequence of β adrenergic receptor activation in this cell model. As a result, these studies represent the first description of an agonist-dependent relationship between a small heat shock protein and β-arrestin to form a previously unknown antiapoptotic “signalosome.”

SSeCKS immunolabeling in rat primary sensory neurons.

SSeCKS (src suppressed C kinase substrate) is a protein kinase C substrate that may play a role in tumor suppression. Recently described in fibroblasts, testes and mesangial cells, SSeCKS may have a function in the control of cell signaling and cytoskeletal arrangement. To investigate the distribution of SSeCKS throughout the nervous system, representative sections of brain, spinal cord and dorsal root ganglia were processed using immunofluorescence. Labeling of central axonal collaterals of primary sensory neurons was observed in the dorsal horn at all spinal levels. SSeCKS-immunoreactivity was also observed in the cerebellum, medulla and sensory ganglia (including trigeminal ganglia). The pattern and distribution of anti-SSeCKS labeling in dorsal root ganglia and the dorsal horn of the spinal cord was similar to that observed for other markers of small primary sensory neurons. Therefore, the coexistence of SSeCKS with substance P, CGRP and acid phosphatase was examined in sections of sensory ganglia, spinal cord and medulla using double immunofluorescent labeling for SSeCKS and substance P/CGRP or sequential SSeCKS immunofluorescence and acid phosphatase/fluoride-resistant acid phosphatase enzyme histochemistry. A small portion of the SSeCKS-labeled cell bodies appeared to represent a subpopulation of substance P (4.8%) and CGRP (4.7%) containing neurons, while 45.0% contained fluoride-resistant acid phosphatase reactivity. These results indicate that SSeCKS has a restricted distribution within the nervous system and that expression of this protein may reflect the specific signaling requirements of a distinct population of nociceptive sensory neurons.

RhoA Kinase (Rock) and p90 Ribosomal S6 Kinase (p90Rsk) phosphorylation of the sodium hydrogen exchanger (NHE1) is required for lysophosphatidic acid-induced transport, cytoskeletal organization and migration☆

The sodium hydrogen exchanger isoform one (NHE1) plays a critical role coordinating asymmetric events at the leading edge of migrating cells and is regulated by a number of phosphorylation events influencing both the ion transport and cytoskeletal anchoring required for directed migration. Lysophosphatidic acid (LPA) activation of RhoA kinase (Rock) and the Ras-ERK growth factor pathway induces cytoskeletal reorganization, activates NHE1 and induces an increase in cell motility. We report that both Rock I and II stoichiometrically phosphorylate NHE1 at threonine 653 in vitro using mass spectrometry and reconstituted kinase assays. In fibroblasts expressing NHE1 alanine mutants for either Rock (T653A) or ribosomal S6 kinase (Rsk; S703A) we show that each site is partially responsible for the LPA-induced increase in transport activity while NHE1 phosphorylation by either Rock or Rsk at their respective site is sufficient for LPA stimulated stress fiber formation and migration. Furthermore, mutation of either T653 or S703 leads to a higher basal pH level and a significantly higher proliferation rate. Our results identify the direct phosphorylation of NHE1 by Rock and suggest that both RhoA and Ras pathways mediate NHE1-dependent ion transport and migration in fibroblasts.

HIF-dependent regulation of AKAP12 (gravin) in the control of human vascular endothelial function

Hypoxia has been widely implicated in many pathological conditions, including those associated with inflammation and tumorigenesis. A number of recent studies have implicated hypoxia in the control of vasculogenesis and permeability, the basis for which is not fully understood. Here we examine the transcriptional regulation of angiogenesis and permeability by hypoxia in endothelial cells. Guided by a global profiling approach in cultured endothelial cells, these studies revealed the selective induction of human gravin (protein kinase A anchoring protein 12) by hypoxia. Analysis of the cloned gravin promoter identified a functional hypoxia‐responsive region including 2 binding sites for hypoxia‐inducible factor (HIF). Site‐directed mutagenesis identified the most distal HIF‐binding site as essential for the induction of gravin by hypoxia. Further studies examining gravin gain and loss of function confirmed strong dependence of gravin in control of microvascular endothelial tube formation, wherein gravin functions as a “braking” system for angiogenesis. Additional studies in confluent endothelia revealed that gravin functionally couples to control endothelial barrier function in response to protein kinase A (PKA) agonists. Taken together, these results demonstrate transcriptional coordination of gravin by HIF‐1α and amplified PKA‐dependent endothelial responses. These findings provide an important link between hypoxia and metabolic conditions associated with inflammation and angiogenesis.—Weissmüller, T., Glover, L. E., Fennimore, B., Curtis, V. F., MacManus, C. F., Ehrentraut, S. F., Campbell, E. L., Scully, M., Grove, B. D., Colgan, S. P. HIF‐dependent regulation of AKAP12 (gravin) in the control of human vascular endothelial function. FASEB J. 28, 256–264 (2014). www.fasebj.org

Receptor-mediated Ca2+ and PKC signaling triggers the loss of cortical PKA compartmentalization through the redistribution of gravin

A-Kinase Anchoring Proteins (AKAPs) direct the flow of cellular information by positioning multiprotein signaling complexes into proximity with effector proteins. However, certain AKAPs are not stationary but can undergo spatiotemporal redistribution in response to stimuli. Gravin, a 300kD AKAP that intersects with a diverse signaling array, is localized to the plasma membrane but has been shown to translocate to the cytosol following the elevation of intracellular calcium ([Ca(2+)]i). Despite the potential for gravin redistribution to impact multiple signaling pathways, the dynamics of this event remain poorly understood. In this study, quantitative microscopy of cells expressing gravin-EGFP revealed that Ca(2+) elevation caused the complete translocation of gravin from the cell cortex to the cytosol in as little as 60s of treatment with ionomycin or thapsigargin. In addition, receptor mediated signaling was also shown to cause gravin redistribution following ATP treatment, and this event required both [Ca(2+)]i elevation and PKC activation. To understand the mechanism for Ca(2+) mediated gravin dynamics, deletion of calmodulin-binding domains revealed that a fourth putative calmodulin binding domain called CB4 (a.a. 670-694) is critical for targeting gravin to the cell cortex despite its location downstream of gravins membrane-targeting domains, which include an N-terminal myristoylation site and three polybasic domains. Finally, confocal microscopy of cells co-transfected with gravin-EYFP and PKA RII-ECFP revealed that gravin redistribution mediated by ionomycin, thapsigargin, and ATP each triggered the gravin-dependent loss of PKA localized at the cell cortex. Our results support the hypothesis that gravin redistribution regulates cross-talk between PKA-dependent signaling and receptor-mediated events involving Ca(2+) and PKC.

Renoprotection from Diabetic Complications in OVE Transgenic Mice by Endothelial Cell Specific Overexpression of Metallothionein: A TEM Stereological Analysis

We previously demonstrated that OVE transgenic diabetic mice are susceptible to chronic complications of diabetic nephropathy (DN) including substantial oxidative damage to the renal glomerular filtration barrier (GFB). Importantly, the damage was mitigated significantly by overexpression of the powerful antioxidant, metallothionein (MT) in podocytes. To test our hypothesis that GFB damage in OVE mice is the result of endothelial oxidative insult, a new JTMT transgenic mouse was designed in which MT overexpression was targeted specifically to endothelial cells. At 60 days of age, JTMT mice were crossed with age‐matched OVE diabetic mice to produce bi‐transgenic OVE‐JTMT diabetic progeny that carried the endothelial targeted JTMT transgene. Renal tissues from the OVE‐JTMT progeny were examined by unbiased TEM stereometry for possible GFB damage and other alterations from chronic complications of DN. In 150 day‐old OVE‐JTMT mice, blood glucose and HbA1c were indistinguishable from age‐matched OVE mice. However, endothelial‐specific MT overexpression in OVE‐JTMT mice mitigated several DN complications including significantly increased non‐fenestrated glomerular endothelial area, and elimination of glomerular basement membrane thickening. Significant renoprotection was also observed outside of endothelial cells, including reduced podocyte effacement, and increased podocyte and total glomerular cell densities. Moreover, when compared to OVE diabetic animals, OVE‐JTMT mice showed significant mitigation of nephromegaly, glomerular hypertrophy, increased mesangial cell numbers and increased total glomerular cell numbers. These results confirm the importance of oxidative stress to glomerular damage in DN, and show the central role of endothelial cell injury to the pathogenesis of chronic complications of diabetes. Anat Rec, 2017.