Direct involvement of protein myristoylation in myristoylated alanine-rich C kinase substrate (MARCKS)-calmodulin interaction.
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.
D000595:Amino Acid Sequence; D002135:Calcium-Binding Proteins; D002147:Calmodulin / Q000378:metabolism*; D005959:Glucosidases; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D009994:Osmolar Concentration; D010750:Phosphoproteins / Q000378:metabolism*; D011485:Protein Binding; D013050:Spectrometry, Fluorescence
D002135:Calcium-Binding Proteins; D002147:Calmodulin; D047908:Intracellular Signaling Peptides and Proteins; C000619710:MARCKS protein, human; D008565:Membrane Proteins; D010750:Phosphoproteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D005959:Glucosidases; C070368:PRKCSH protein, human