Differential and Regulated Binding of cAMP-dependent Protein Kinase and Protein Kinase C Isoenzymes to Gravin in Human Model Neurons

Abstract

The membrane cortex has an important role in generating and maintaining spatially and functionally distinct domains in neurons. As a tool to functionally characterize molecules of the membrane cortex, we generated novel monoclonal antibodies against a fraction enriched for components of the neuronal membrane skeleton. We obtained two antibodies against the kinase-anchoring protein gravin. Gravin was strongly up-regulated during differentiation of human model neurons (NT2-N neurons) and was enriched at the inner peripheral cortex in close proximity to the plasma membrane where its localization primarily depended on association with membranes. In differentiated neurons, gravin colocalized in putative signaling complexes with protein kinase C (PKC␤II) and partially with PKC␣ and cAMP-dependent protein kinase (PKA). Colocalization with PKC⑀ was not observed. PKC␤II, PKC␣, and PKA but not PKC⑀ coprecipitated with gravin indicating physical interaction. Binding of gravin to PKC␣ required the presence of Ca 2؉ and was increased after inhibition of PKC. In contrast, binding of PKC␤II and PKA were independent of Ca 2؉ and PKC inhibition. Activation of PKC decreased binding of PKC␣ to gravin, decreased its association with the plasma membrane, and reduced the mean size of gravin particles. Taken together the data suggest that gravin provides a dynamic platform to localize kinases in an isoenzyme-specific and activation-dependent manner at specific sites in neurons.During development, many neurons establish a complex morphology that is characterized by the formation of multiple and in many cases highly branched neurites. Within the neurites, many organelles and proteins exhibit a nonuniform distribution that is thought to provide the basis for the formation of structurally and functionally distinct domains. For example, mitochondria are clustered at sites of high energy consumption, and several cytoskeleton-associated proteins are enriched at positions where active process outgrowth occurs. Some kinases and phosphatases also show a nonuniform distribution in neurons and are enriched at specific regions. In many cases this is achieved by locally anchoring them to cytosolic filament systems or to components of the cortical membrane skeleton.

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

“…A role for PS in facilitating PKC/SSeCKS association in cells cannot be ruled out, however, because we show reciprocal co-IP between SSeCKS and various cPKC or nPKC family members from cell lysates, which likely contain physiologic levels of free PS (non-membranous). This agrees with previous findings from Piontek and Brandt (36) in which endogenous human SSeCKS (GRAVIN) and PKC␣, -␤I, and -␤II could be co-immunoprecipitated from lysates of NT2-N1 neurons without the addition of PS.…”

“…The aa 745-753-containing domain bound all three preparations of PKC␣ we tested irrespective of kinase activity. This domain also bound a commercial enzymatically active preparation of PKC⑀, a finding that contrasts with a previous report (36), although it should be noted that our conditions are at saturating levels. In contrast, enzymatically inactive preparations of PKC␣ failed to bind to the aa 596 -605-containing domain, whereas the enzymatically active commercial PKC␣ preparation bound this domain.…”

“…We also showed (32) that the PS-independent co-IP of SSeCKS and PKC could be enhanced by the addition of high levels of PS (123 M). In contrast, Piontek and Brandt (36) showed that the ability of GRAVIN (the human SSeCKS/ AKAP12 orthologue) to co-immunoprecipitate PKC␣ or -␤II was not enhanced by PS. Thus, we investigated whether SSeCKS encodes distinct PS-dependent and -independent binding activities and if so whether these were encoded by different SSeCKS protein domains.…”

“…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.…”

AKAPs (A-kinase anchoring proteins) and molecules that compose their G-protein-coupled receptor signalling complexes

“…Recently, it has been shown in human model neurons (NT2-N neurons) that PKCα and PKCβII can differentially interact with AKAP250, the former requiring the presence of Ca 2+ for docking, whereas the binding of the latter (as well as PKA) was independent of Ca 2+ . Inhibition of PKC activity increased PKCα binding, but did not affect that of PKCβII [46]. Thus overlap of docking sites, as well as changes in local concentration of Ca 2+ , may dictate the character of the composition and activities of AKAP-based signalling complexes for GPCRs.…”