Identification of the Calmodulin-binding Domain of Neuron-specific Protein Kinase C Substrate Protein CAP-22/NAP-22

Abstract

Various proteins in the signal transduction pathways as well as those of viral origin have been shown to be myristoylated. Although the modification is often essential for the proper functioning of the modified protein, the mechanism by which the modification exerts its effects is still largely unknown. Brain-specific protein kinase C substrate, CAP-23/NAP-22, which is involved in the synaptogenesis and neuronal plasticity, binds calmodulin, but the protein lacks any canonical calmodulin-binding domain. In the present report, we show that CAP-23/NAP-22 isolated from rat brain is myristoylated and that the modification is directly involved in its interaction with calmodulin. Myristoylated and non-myristoylated recombinant proteins were produced in Escherichia coli, and their calmodulin-binding properties were examined. Only the former bound to calmodulin. Synthetic peptides based on the N-terminal sequence showed similar binding properties to calmodulin, only when they were myristoylated. The calmodulin-binding site narrowed down to the myristoyl moiety together with a nine-amino acid N-terminal basic domain. Phosphorylation of a single serine residue in the N-terminal domain (Ser 5 ) by protein kinase C abolished the binding. Furthermore, phosphorylation of CAP-23/NAP-22 by protein kinase C was also found myristoylation-dependent, suggesting the importance of myristoylation in protein-protein interactions.

The binding of myristoylated N‐terminal nonapeptide from neuron‐specific protein CAP‐23/NAP‐22 to calmodulin does not induce the globular structure observed for the calmodulin—nonmyristoylated peptide complex

“…In fact, a recent report describes an NMR structure for a complex between a peptide from Ca 2ϩ pump and CaM which differs from that of the MLCK peptide complex in that it does not assume a collapsed globular structure~Elshorst et al, 1999!. It cannot be assumed that the mC0N9 and Ca 2ϩ pump peptides bind to CaM in the same manner because mC0N9 is unlikely to form a helical structurẽ Takasaki et al, 1999! differently from the Ca 2ϩ pump that takes a helical structure in solution.…”

Protein-Ligand Interactions and Allosteric Regulation of Activity in DREAM Protein

“…Our observation that DREAM associates with DNA independent of calcium is in contrast to previous work (Osawa et al 2005 (Deisseroth, Heist & Tsien 1998, Zaidi et al 2004. It is also possible that posttranslational modification such as myristoylation/palmytoylation at Cys45/Cys46 could enhance the affinity between CaM and DREAM and decrease the observed EC50, an effect which has been observed in other CaM binding proteins (Takasaki et al 1999). …”

Direct Involvement of Protein Myristoylation in Myristoylated Alanine-rich C Kinase Substrate (MARCKS)-Calmodulin Interaction

“…However, the involvement of the protein myristoylation in protein-protein interaction has never been clearly demonstrated, although the issue has been the subject of extensive studies (11)(12)(13). Recently, we have shown that the modification is directly involved in the interaction of a brainspecific protein kinase C substrate, CAP-23/NAP-22, with calmodulin (14,15). Furthermore, the phenomena is observed in the binding HIV-1 Nef with calmodulin (16).…”

“…The peptides were purified by reversed-phase high-performance liquid chromatography (HPLC) using a C18 column (Waters, Bondasphere 5 C18 -300Å, 1.9 ϫ 15 cm). They were judged to be of greater than 95% purity by analytical HPLC and electrospray mass spectrometry (14,25). Peptide concentration was determined by quantitative amino acid analysis.…”