Gravin dynamics regulates the subcellular distribution of PKA

Abstract

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.

FRET biosensors reveal AKAP-mediated shaping of subcellular PKA activity and a novel mode of Ca2+/PKA crosstalk

“…Gravin-EGFP, gravin-V5/His, (ΔCB4) gravin-EGFP, and PKA RII-ECFP were constructed and described previously [7, 9]. This study utilized four additional gravin constructs which were made as follows: A mutant gravin construct termed “(mutCB4) gravin-EGFP” was generated with three substitutions in the CB4 domain which replaced valine 672, valine 676, and leucine 681 each with alanine (V672A, V676A, L681A) using site-directed mutagenesis (Phusion®, New England Biolabs, product F-541; forward primer 5′ C ATCTTGGGAAGCT GC AATTTGTGTG; reverse primer 5′CTGAGGTATCC G CCTT T CGCTTTGGT; mutagenic nucleotides underlined).…”

“…In response to either PKC activation or intracellular calcium ([Ca 2+ ] i ) elevation, gravin is redistributed away from the membrane along with PKA that is bound to gravin. Gravin redistribution by PKC activation was shown by Yan et al [7] to redirect gravin and PKA to a juxtanuclear vesicular compartment. In response to [Ca 2+ ] i elevation, Tao et al [8] showed that gravin redistributes to the cytosol through a mechanism thought to involve Ca 2+ /calmodulin binding to gravin’s membrane-associated polybasic domains, PB1-3.…”

Control of Protein Kinase C Activity, Phorbol Ester-induced Cytoskeletal Remodeling, and Cell Survival Signals by the Scaffolding Protein SSeCKS/GRAVIN/AKAP12

“…Given that SSeCKS is an in vitro and in vivo PKC substrate (26,32,44) and that short term PMA treatment induces a co-translocation of SSeCKS and actin fibers to the perinucleus (32,36,45), we addressed whether KO-MEF were defective in PMA-induced PKC translocation, cytoskeletal remodeling, and cell rounding. Although KO-MEF exhibit increased PMA-induced PKC activation (Fig.…”

Activity-dependent inhibitory gating in molecular signaling cascades induces a novel form of intermediate-term synaptic facilitation in Aplysia californica

“…AKAP-RII subcellular localization can be changed by Ca 2+ and PKC, notably from the plasma membrane to the cytoplasm (Yan et al 2009;Schott and Grove 2013). This redistribution, in Figure 4.…”