MARCKS Is a Natively Unfolded Protein with an Inaccessible Actin-binding Site

Abstract

Myristoylated alanine-rich C kinase substrate (MARCKS) is an unfolded protein that contains well characterized actin-binding sites within the phosphorylation site domain (PSD), yet paradoxically, we now find that intact MARCKS does not bind to actin. Intact MARCKS also does not bind as well to calmodulin as does the PSD alone. Myristoylation at the N terminus alters how calmodulin binds to MARCKS, implying that, despite its unfolded state, the distant N terminus influences binding events at the PSD. We show that the free PSD binds with site specificity to MARCKS, suggesting that long-range intramolecular interactions within MARCKS are also possible. Because of the unusual primary sequence of MARCKS with an overall isoelectric point of 4.2 yet a very basic PSD (overall charge of ؉13), we speculated that ionic interactions between oppositely charged domains of MARCKS were responsible for long-range interactions within MARCKS that sterically influence binding events at the PSD and that explain the observed differences between properties of the PSD and MARCKS. Consistent with this hypothesis, chemical modifications of MARCKS that neutralize negatively charged residues outside of the PSD allow the PSD to bind to actin and increase the affinity of MARCKS for calmodulin. Similarly, both myristoylation of MARCKS and cleavage of MARCKS by calpain are shown to increase the availability of the PSD so as to activate its actin-binding activity. Because abundant evidence supports the conclusion that MARCKS is an important protein in regulating actin dynamics, our data imply that post-translational modifications of MARCKS are necessary and sufficient to regulate actin-binding activity. Myristoylated alanine-rich C kinase substrate (MARCKS)1 is a well characterized, charge-polarized, natively unfolded molecule (1-3) with a centrally located active site known as the phosphorylation site domain (PSD). Consistent with the paradigm for natively unfolded proteins, MARCKS is thought to interact with several ligands so as to integrate information from various signal transduction pathways to produce an output signal that regulates cell motile and contractile function. Numerous studies of the MARCKS protein have utilized a peptide with a sequence that corresponds to the PSD peptide as a substitute for studying interactions between the intact protein and its multiple ligands (3, 4). Although this approach intuitively appears to be logical, given the unfolded state of the native protein, the substitution of the PSD peptide for the intact protein has never been rigorously justified. In fact, there are several reported experiments that imply that the PSD peptide behaves differently from intact MARCKS. The nonphosphorylated PSD peptide is known to have extended structure, to nucleate polymerization, and to cross-link F-actin filaments (5-7), presumably because of two binding sites with a site-specific K d of ϳ0.5 M for F-actin (8). Although the PSD of MARCKS and its homolog MARCKS-related protein have both been shown to bind to actin with si...

Two N-Myristoyltransferase Isozymes Play Unique Roles in Protein Myristoylation, Proliferation, and Apoptosis

“…The myristoylated form of MARCKS, which migrates with an apparent molecular weight of 80 kDa, is clearly reduced in the NMT1 knockdown cells and barely visible in the double knockdown cells. MARCKS can be cleaved by calpain to produce a fragment that migrates with an apparent molecular weight of 40 kDa, and this cleavage has been shown to increase the rate of MARCKS-induced polymerization of actin (38). Levels of the p40 MARCKS product were also reduced in the NMT1 knockdown and double knockdowns.…”

TRPC1 regulates skeletal myoblast migration and differentiation

“…Dulong and colleagues demonstrated the direct cleavage of MARCKS by calpain and its involvement in myoblast migration (Dulong et al, 2004). Calpain-dependent MARCKS proteolysis was also shown to activate its actin binding activity and therefore its ability to modulate actin dynamics and cell migration (Tapp et al, 2005). This proteolysis seems to be PKCα dependent (Goudenege et al, 2005).…”

Synaptopodin family of natively unfolded, actin binding proteins: physical properties and potential biological functions

“…Several actin binding proteins contain large unstructured regions. Those proteins include caldesmon (Permyakov et al 2003), MARCKS (myristoylated alaninerich C kinase substrate) (Tapp et al 2005), ARG (Abl-related only the composition of the proteins, therefore non-coding exons 1 and 3 in synaptopodin are not shown. Except for the unnamed protein that is listed with the NCBI accession number BAG65020 the isoforms of each subfamily share a common core region gene) (Buffa et al 2007), and dematin (Chen et al 2009).…”