Studies on the scaffolding properties of MAP2, TAKAP80 and SSeCKS

Abstract

The cAMP-dependent protein kinase (PKA) is involved in the regulation of key cellular processes including gene expression, metabolism and cytoskeletal reorganization. These diverse responses result from the ability of PKA to selectively phosphorylate different sets of cellular proteins. Targeting of PKA to specific subcellular structures plays an essential role in substrate selectivity as shown by in vivo disruption of PKA targeting. Targeting is mediated by A-kinase anchoring proteins (AKAPs) such as MAP2, TAKAP80 and SSeCKS, among others. (1) In neurons PKA is targeted to microtubules though binding of RIIbeta to amino acid residues 86--103 of MAP2, a region predicted to form an amphipathic alpha-helix, the proposed RII-binding motif of AKAPs. NMR studies on recombinant MAP2(72--110) showed that the binding motif does not have an alpha-helical conformation in solution. Instead, MAP2(72--110) adopts an helical conformation upon binding to RIIbeta suggesting that the helical motif can be formed dynamically. This flexibility may represent a potential mechanism to control PKA targeting. (2) Phosphorylation of RIIbeta at Thr69 by cyclin-dependent kinases may have a role in the modulation of PKA targeting in the mammalian brain. Thr69 is located in the vicinity of the AKAP binding domain. (3) Both MAP2 and TAKAP80, highly abundant proteins in neurons and sperm respectively, contain a proline-rich (PR) sequence with multiple PXXP repeats. We found that the PR region of MAP2 interacts with Hsc70 (heat-shock cognate protein) and N-WASP (neural Wiskott-Aldrich syndrome protein). These proteins are involved in neural plasticity phenomena. TAKAP80 was found to interact with Grp78 (glucose-regulated protein 78), Hsp70 (heat-shock protein 70) and PP1c (protein phosphatase 1c) in testis. We suggest that the fibrous sheath TAKAP80 co-localizes PKA and PP1 to this structure to participate in sperm motility control. Interestingly, none of the proteins identified contains SH3 domains. (4) SSeCKS, an AKAP and a PKC-binding/substrate protein, is highly expressed in rat testis. Our studies show that the expression of SSeCKS in rat testis increases with age reaching its maximum in the adult. The protein is present exclusively in post-meiotic developing spermatids with the highest levels in elongating spermatids. We suggest that PKA and SSeCKS may be involved in the morphological changes of spermiogenesis.