Targeted protein kinase A and PP-2B regulate insulin secretion through reversible phosphorylation.

Protein kinases and phosphatases play key roles in integrating signals from various insulin secretagogues. In this study, we show that the activities of the cAMP-dependent protein kinase (PKA) and the calcium/calmodulin-dependent phosphatase, PP-2B are coordinated resulting in the regulation of insulin secretion. Transient inhibition of PP-2B, using the immunosuppressant FK506, increased forskolin stimulated insulin secretion by 2.5-fold +/- 0.3 (n = 6) in rat islets and RINm5F cells. Surprisingly, forskolin treatment resulted in the dephosphorylation of the vesicle-associated protein synapsin 1 and increased PP-2B activity by 2.98 +/- 0.97-fold (n = 4). One potential explanation for the observed coordination of PKA and PP-2B activity is their colocalization through a mutual anchoring protein, AKAP79/150. Accordingly, RINm5F cells expressing AKAP79 exhibited decreased insulin secretion, reduced PP-2B activity and were insensitive to FK506. This suggests that AKAP targeting of PKA and PP-2B maintains a signal transduction complex that may regulate reversible phosphorylation events involved in insulin secretion.

A novel lipid-anchored A-kinase Anchoring Protein facilitates cAMP-responsive membrane events.

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 Ca2+ channel promoted a 34 9% increase in cAMP-responsive Ca2+ currents. In contrast, a targeting-deficient mutant of AKAP18 had no effect on Ca2+ currents in response to the application of a cAMP analog. Further studies demonstrate that AKAP18 facilitates GLP-1-mediated insulin secretion in a pancreatic beta cell line (RINm5F), suggesting that membrane anchoring of the kinase participates in physiologically relevant cAMP-responsive events that may involve ion channel activation.

Anchoring and scaffold proteins for kinases and phosphatases.

Many hormones mediate their intracellular actions by triggering signal transduction pathways that alter the phosphorylation state of key regulatory proteins. Protein phosphorylation is a reversible process involving two classes of signaling enzymes: protein kinases, which catalyze the transfer of phosphate from ATP onto substrate proteins, and phosphoprotein phosphatases, which perform the dephosphorylation step. To insure tight control of hormonally initiated phosphorylation events, the activity of multifunctional kinases and phosphatases is precisely regulated and responds to fluctuations in diffusible second messengers such as Ca2+, phospholipid, and cAMP. Another mechanism that contributes to their regulation is to restrict the location of these enzymes to certain subcellular compartments. Subcellular targeting enhances the selectivity of serine/threonine phosphatases and kinases by favoring their accessibility to certain substrate proteins. Compartmentalization is achieved through a "targeting moiety," which is defined as that part of a phosphatase or kinase that directs the catalytic subunit to a certain subcellular environment. The targeting moiety restricts the location of a phosphatase or kinase through association with a "targeting locus." These are often structural membrane proteins, cytoskeletal components, or cellular organelles. Targeting subunits for the type I phosphatase and protein kinase C have been identified; however, the focus of this chapter centers around a family of anchoring proteins, called AKAPs, that localize the type II cAMP-dependent protein kinase (PKA). Structure-function analysis suggest that each anchoring protein binds to the RII dimer through a conserved amphipathic helix region and is tethered to specific subcellular sites via association of a targeting domain with structural proteins or cellular organelles. Peptides patterned after the amphipathic region have been used to probe the functional significance of PKA anchoring inside cells and have begun to be established by that disruption RII/AKAP interaction in vivo has concomitant effects on certain PKA-mediated phosphorylation events. In addition, multivalent binding proteins such as AKAP79 and AKAP250 have been characterized and appear to serve as platforms for the assembly of kinase/phosphatase signaling complexes. Collectively, these studies suggest that the AKAPs represent a growing family of regulatory proteins that provide a molecular architecture that organizes the intracellular location of a single or multiple multifunctional kinase.

Anchoring of protein kinase A facilitates hormone-mediated insulin secretion.

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 cAMP-mediated elevation of intracellular calcium suggesting that localized PKA phosphorylation events augment calcium flux.

Cloning and characterization of a novel A-kinase anchoring protein. AKAP 220, association with testicular peroxisomes.

Compartmentalization of the type II cyclic AMP-dependent kinase (PKA) is achieved through association of the regulatory subunit (RII) with A-kinase anchoring proteins (AKAPs). Using an interaction cloning strategy with RIIalpha as a probe, we have isolated cDNAs encoding a novel 1129-amino acid protein that contains both a PKA binding region and a peroxisome targeting motif. Northern analysis detected mRNAs of 9.7 and 7.3 kb in several rat tissues with the highest levels present in the brain and testis. Western analysis and RII overlay experiments showed that the protein is approximately 220 kDa and was, therefore, named AKAP 220. Immunoprecipitation of AKAP 220 from rat testis extracts resulted in co-purification of the type II PKA holoenzyme. The specific activity of PKA increased 458-fold from 7.2 pmol/min/mg in the cell lysate to 3.3 nmol/min/mg in the immunoprecipitate. Immunohistochemical analysis of rat testicular TM4 cells showed that AKAP 220 and a proportion of RII were co-localized in microbodies that appear to be a subset of peroxisomes. Collectively, these results suggest that AKAP 220 may play a role in targeting type II PKA for cAMP-responsive peroxisomal events.

Occupational therapy personnel and AIDS: attitudes, knowledge, and fears.

This study explored the attitudes, knowledge, and fears of occupational therapists and certified occupational therapy assistants regarding AIDS and HIV. The 119 respondents' scores related to knowledge and fear revealed that many had significant fears about AIDS, which in turn may inhibit their willingness to care for persons with AIDS. The respondents also indicated a need for specific information about the condition, including current research data and information on infection control.

Nursing students' attitudes toward AIDS.

A study of 177 baccalaureate nursing students was conducted to explore their knowledge, fears, beliefs and other attitudes regarding AIDS. Lazarus' theory related to coping with threatening events provided the theoretical framework. Students with a high fear score were less willing to care for AIDS patients, had higher knowledge scores, and were more homophobic. While 96.6% of the students felt that AIDS patients are entitled to the same care as any other patient, 49% preferred not to care for AIDS patients. Thirty-six percent thought nursing students should not be assigned to care for AIDS patients. Most of the students (70.6%) got their information about AIDS from the media. Nursing faculty must respond by including current, correct information when instructing students about AIDS. Faculty also need to provide opportunities for students to ask questions and share their fears regarding AIDS.