Phosphorylation by cAMP-dependent protein kinase (PKA) increases the activity of class C L-type Ca 2؉ channels which are clustered at postsynaptic sites and are important regulators of neuronal functions. We investigated a possible mechanism that could ensure rapid and efficient phosphorylation of these channels by PKA upon stimulation of cAMP-mediated signaling pathways. A kinase anchor proteins (AKAPs) bind to the regulatory R subunits of PKA and target the holoenzyme to defined subcellular compartments and substrates. Class C channels isolated from rat brain extracts by immunoprecipitation contain an endogenous kinase that phosphorylates kemptide, a classic PKA substrate peptide, and also the main phosphorylation site for PKA in the pore-forming ␣ 1 subunit of the class C channel complex, serine 1928. The kinase activity is inhibited by the PKA inhibitory peptide PKI(5-24) and stimulated by cAMP. Physical association of the catalytic C subunit of PKA with the immunoisolated class C channel complex was confirmed by immunoblotting. A direct protein overlay binding assay performed with 32 P-labeled RII␤ revealed a prominent AKAP with an M r of 280,000 in class C channel complexes. The protein was identified by immunoblotting as the microtubule-associated protein MAP2B, a well established AKAP. Class C channels did not contain tubulin and MAP2B association was not disrupted by dilution or addition of nocodazole, two treatments that cause dissociation of microtubules. In vitro experiments show that MAP2B can directly bind to the ␣ 1 subunit of the class C channel. Our findings indicate that PKA is an integral part of neuronal class C L-type Ca 2؉ channels and suggest that the AKAP MAP2B may mediate this interaction. Neither PKA nor MAP2B were detected in immunoprecipitates of ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid-type glutamate receptors or class B N-type Ca 2؉ channels. Accordingly, MAP2B docked at class C Ca 2؉ channels may be important for recruiting PKA to postsynaptic sites.

We have cloned cDNA that encodes six novel A kinase anchor proteins (collectively named AKAP-KL). AKAP-KL diversity is generated by alternative mRNA splicing and utilization of two translation initiation codons. AKAP-KL polypeptides are evident in lung, kidney, and cerebellum, but are absent from many tissues. Different isoforms predominate in different tissues. Thus, AKAP-KL expression is differentially regulated in vivo. All AKAP-KL isoforms contain a 20-residue domain that avidly binds (K d ϳ 10 nM) regulatory subunits (RII) of protein kinase AII and is highly homologous with the RII tethering site in neuronal AKAP75. The distribution of AKAP-KL is strikingly asymmetric (polarized) in situ. Anchor protein accumulates near the inner, apical surface of highly polarized epithelium in tubules of nephrons. Both RII and AKAP-KL are enriched at an intracellular site that lies just below the plasma membrane of alveolar epithelial cells in lung. AKAP-KL interacts with and modulates the structure of the actin cytoskeleton in transfected cells. We also demonstrate that the tethering domain of AKAP-KL avidly ligates RII subunits in intact cells. AKAP-KL may be involved in (a) establishing polarity in signaling systems and (b) physically and functionally integrating PKAII isoforms with downstream effectors to capture, amplify, and precisely focus diffuse, trans-cellular signals carried by cAMP. Protein kinase A (PKA)1 mediates actions of hormones and neurotransmitters that activate adenylate cyclase (1-4). Signals carried by cAMP are often directed to effectors that accumulate at discrete intracellular sites (5-7). Targeting of signals to these sites can be achieved by generating a non-uniform distribution of PKA molecules. This occurs when PKAII␣ and II␤ isoforms are attached to cytoskeleton or organelles by A kinase anchor proteins (AKAPs) (5, 6). Prototypic anchor proteins (AKAPs 75, 79, and 150) have a binding site for regulatory (RII) subunits of PKAII isoforms and distinct domains that mediate non-covalent coupling of AKAP⅐PKAII complexes to the microtubule-based dendritic cytoskeleton of neurons and the cortical actin cytoskeleton of non-neuronal cells (5-13). Both cytoskeletal locations are closely apposed to the plasma membrane. Thus, anchored PKAII is positioned near a signal generator (adenylate cyclase) and multiple PKA substrate/effector proteins (e.g. myosin light chain kinase, microtubuleassociated protein-2, ion channels, serpentine receptors that couple with G s ) (5,6,14). Effector proteins in cAMP signaling pathways can also be associated with organelles, specialized regions of plasma membrane, or sites in cytoskeleton that are separated from adenylate cyclase by substantial distances. Novel RII-binding proteins apparently participate in the assembly of "distal signaling modules" that are associated with mitochondria, Golgi membranes, peroxisomes, and centrioles (5, 6, 15, 16).Anchored PKAII isoforms may be essential for dissemination of cAMP signals in highly polarized epithelium. Epithelial cells of l...

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