Compartmentalization of protein kinases with substrates is a mechanism that may promote specificity of intracellular phosphorylation events. We have cloned a low-molecular weight A-kinase Anchoring Protein, called AKAP18, which targets the cAMP-dependent protein kinase (PKA) to the plasma membrane, and permits functional coupling to the L-type calcium channel. Membrane anchoring is mediated by the first 10 amino acids of AKAP18, and involves residues Gly1, Cys4 and Cys5 which are lipid-modified through myristoylation and dual palmitoylation, respectively. Transient transfection of AKAP18 into HEK-293 cells expressing the cardiac L-type Ca 2⍣ channel promoted a 34 ⍨ 9% increase in cAMP-responsive Ca 2⍣ currents. In contrast, a targeting-deficient mutant of AKAP18 had no effect on Ca 2⍣ currents in response to the application of a cAMP analog. Further studies demonstrate that AKAP18 facilitates GLP-1-mediated insulin secretion in a pancreatic β cell line (RINm5F), suggesting that membrane anchoring of the kinase participates in physiologically relevant cAMP-responsive events that may involve ion channel activation.

Impaired insulin secretion is a characteristic of non-insulin-dependent diabetes mellitus (NIDDM). One possible therapeutic agent for NIDDM is the insulinotropic hormone glucagon-like peptide 1 (GLP-1). GLP-1 stimulates insulin secretion through several mechanisms including activation of protein kinase A (PKA). We now demonstrate that the subcellular targeting of PKA through association with A-kinase-anchoring proteins (AKAPs) facilitates GLP-1-mediated insulin secretion. Disruption of PKA anchoring by the introduction of anchoring inhibitor peptides or expression of soluble AKAP fragments blocks GLP-1 action in primary islets and cAMP-responsive insulin secretion in clonal beta cells (RINm5F). Displacement of PKA also prevented cAMPmediated elevation of intracellular calcium suggesting that localized PKA phosphorylation events augment calcium f lux.Subcellular targeting of protein kinases and phosphatases is an efficient mechanism to selectively regulate cellular events by ensuring that phosphorylation only occurs at precise intracellular sites (1-3). Accumulating evidence suggests that the subcellular location of the cAMP-dependent protein kinase (PKA) is a determinant in the preferential phosphorylation of certain PKA substrates (4, 5). Compartmentalization of the PKA holoenzyme is conferred by the association of the regulatory subunit dimer (RII) with a family of A-kinaseanchoring proteins (AKAPs) (6, 7). Anchoring not only places the kinase close to preferred substrates but also positions the PKA holoenzyme at sites where it can optimally respond to fluctuations in the second messenger cAMP (1-3). This higher order of spatial organization may also coordinate effectormediated events that integrate second messenger signals (8-11). For example, insulin secretion from pancreatic beta cells requires the coordinate action of metabolites, hormones, and neurotransmitters (12, 13). A recently identified hormone, glucagon-like peptide 1 (GLP-1), potentiates glucosemediated insulin secretion through activation of PKA (14-16).In this report we demonstrate that the correct subcellular location of PKA is a determinant in the cAMP signaling pathway in response to GLP-1 to induce insulin secretion from pancreatic islets and related cell lines. METHODRII Overlay. The presence of AKAPs in primary rat pancreas was detected by a solid phase RII overlay as previously described (17, 18). Fifty micrograms of total pancreas protein was separated by electrophoresis on an SDS͞10% polyacrylamide gel and electrotransferred to nitrocellulose. Filters were pretreated with either 3 M Ht31 peptide or 3 M Ht31P peptide and then incubated with 32 P-labeled RII␣ overnight at room temperature. RII binding proteins were detected by autoradiography.Preparation of Primary Islets and Transfected RINm5F Cells. Normal rat pancreas was isolated and infused with Hanks' buffer before dissection. Pancreatic islets were isolated by collagenase digestion and plated on Falcon tissue culture dishes. Islets were maintained in culture for up to 5 day...