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

Abstract

MARCKS, a major in vivo substrate of protein kinase C, interacts with plasma membranes in a phosphorylation-, myristoylation-, and calmodulin-dependent manner. Although we have previously observed that myristoylated and non-myristoylated MARCKS proteins behave differently during calmodulin-agarose chromatography, the role of protein myristoylation in the MARCKS-calmodulin interaction remained to be elucidated. Here we demonstrate that the myristoyl moiety together with the N-terminal protein domain is directly involved in the MARCKS-calmodulin interaction. Both myristoylated and non-myristoylated recombinant MARCKS bound to calmodulin-agarose at low ionic strengths, but only the former retained the affinity at high ionic strengths. A quantitative analysis obtained with dansyl (5-dimethylaminonaphthalene-1-sulfonyl)-calmodulin showed that myristoylated MARCKS has an affinity higher than the non-myristoylated protein. Furthermore, a synthetic peptide based on the N-terminal sequence was found to bind calmodulin only when it was myristoylated. Only the N-terminal peptide but not the canonical calmodulin-binding domain showed the ionic strength-independent calmodulin binding. A mutation study suggested that the importance of the positive charge in the N-terminal protein domain in the binding.Since the discovery of a covalent-bound myristoyl group in the catalytic subunit of cAMP-dependent protein kinase (1), many proteins have been found to be modified not only with myristoyl group but also with a variety of fatty acids (2-4). Interestingly, the vast majority of acylated proteins are either proteins involved in cellular signaling or those of viral origin. Protein acylation is often essential for the proper functioning of these proteins (5), although in the mechanism by which the modification exerts the effect is largely unknown. Of various kinds of acylation, myristoylation has been implied in the reversible membrane association due to its intermediate hydrophobicity (3, 6). Studies from our own and other laboratories (7,8) have established that such a mechanism is, in fact, operative in the phosphorylation-dependent interaction of MARCKS 1 (myristoylated alanine-rich C kinase substrate) with membranes. In the case of recoverin, the binding of Ca 2ϩ induces a drastic conformational change of the protein, and the myristoyl group hidden inside the protein will protrude from the protein and can interact with membrane (9). The modification has also been shown to affect the protein stability of cAMP-dependent protein kinase (10). 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 (11-13). 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 acyl chain interacts with specifically with...

Binding of Calmodulin to the HIV-1 Matrix Protein Triggers Myristate Exposure

“…Additional electrostatic interactions were also shown to stabilize CaM-protein interactions (44,45,50,51). However, the importance and relative contribution of hydrophobic versus electrostatic factors in myr(ϩ)MA⅐CaM interactions are not known, especially that the hydrophobic myr group can potentially play a role as was observed in other CaM-protein interactions (44,45,50,51). ITC has been used to determine the affinity and thermodynamic parameters of CaM binding to myr(ϩ)MA.…”

“…Myristate Group of MA Is Not Required for CaM BindingSeveral myristoylated proteins including MARCKS, CAP-23/ NAP-22, and HIV-1 Nef were found to bind CaM to facilitate their intracellular localization and membrane targeting (44,45,50,51). Our ITC data described above show that CaM binding to myr(ϩ)MA is entropically driven, indicating a major contribution of hydrophobic contacts.…”

“…Recent studies have shown that myr(Ϫ)MA and short peptides derived from the MA protein interact directly with CaM (48,49). However, it is well established that several myristoylated proteins like the myristoylated alanine-rich C kinase substrate (MARCKS), brain-specific protein kinase C substrate (CAP-23/NAP-22), and HIV-1 Nef interact with CaM via the myr group to facilitate their intracellular localization and membrane targeting (44,45,50,51). Thus, we seek to determine whether the myr group of MA is important for CaM binding and/or whether binding of CaM to MA alters the myr switch mechanism in vitro.…”

AKAPs (A-kinase anchoring proteins) and molecules that compose their G-protein-coupled receptor signalling complexes

“…Although this region is poorly understood in AKAPs, the MARCKS protein-like domain has the potential for providing strong binding to membrane phospholipids, may sequester PtdIns(4,5)P 2 , and, as mentioned above, is certainly a putative binding site of CaM influenced by protein N-myristoylation of MARCKS protein [36]. In AKAP79, the sequence K 31 ASMLC-FKRRKKAAKALKPKAG 52 (where the bold residues represent the dibasic residue motif) also has been implicated as a CaMbinding domain, as well as a weak PKCβII-binding site [37].…”

Differential interaction of β2e with phosphoinositides: A comparative study between β2e and MARCKS

“…2C, D, and E). Considering that N-terminal myristoylation of MARCKS is partially involved in membrane binding, 25 such lipidation may prevent membrane dissociation of the protein from enzymatic activity. However, in cells treated with myristoylation inhibitor 2-hydroxymyristic acid (2-HM), the parallel experiments performed under the same condition showed that de-myristoylation did not affect the location of MARCKS (Fig.…”