Definition
The myristoylated, alanine-rich C kinase substrate (MARCKS) is a major, specific substrate of protein kinase C (PKC) that is phosphorylated during macrophage and neutrophil activation, growth factor-dependent mitogenesis and neurosecretion.
Discovery
In 1982 it was demonstrated that PKC regulates the phosphorylation of an 87 kDa substrate in brain synaptosomes, and that this phosphorylation can be inhibited by calmodulin (CaM)1. 87 kDa protein known today as MARCKS. There are two known members of the MARCKS family: MARCKS, a 32 kDa ubiquitously expressed protein, and MARCKS-related protein (MRP, also known as Mac- MARCKS, F52 or MLP), a 20 kDa protein expressed mainly in brain, reproductive tissues and macrophages 2, 3. MARCKS has been found in Torpedo californica, Xenopus, chicken, mice, rat, cow and human.
Structural Characteristics
MARCKS proteins possess three highly conserved regions. The N-terminus represents a consensus sequence for myristoylation, a co-translational lipid modification attaching myristic acid; the C14 saturated fatty acid, via an amide bond to the amino group of the N-terminal glycine residue. The MH2 domain resembles the cytoplasmic tail of the cation-independent mannose-6-phosphate receptor and is also the site of the only intron-splicing event. Finally, the phosphorylation site domain contains all serine residues known to be PKC phosphorylation sites. This domain has been shown to be central to the function of MARCKS proteins, and is therefore called the effector domain (ED). The ED is highly basic, in contrast with the rest of the protein. MARCKS binds to membranes because these basic residues interact electrostatically with acidic lipids and the myristate inserts hydrophobically into the core of the membrane. Neither of these two interactions on its own is sufficient for significant membrane binding 4, 5.
Electron microscopy has shown rod-shaped, elongated molecules with dimensions of approximately 4.5 nm x 36 nm for MARCKS 32 kDa, while analytical ultracentrifugation measurements gave dimensions of approximately 2 nm x 13 nm for unmyristoylated MRP 20 kDa. CD studies of recombinant MARCKS proteins revealed a high percentage of unfolded sequence, together with a-helical and very few ß-sheet regions in the protein2, 6, 7.
Mode of Action
MARCKS interacts with the plasma membrane of macrophages, neurons and fibroblasts. Phosphorylation by PKC (which attaches negatively charged phosphate groups to the serine residues) abrogates membrane binding of MARCKS in many cell types, since neutralization of the positive charges of the basic residues by the phosphoserine residues abolishes the electrostatic contribution of the ED to membrane binding, and myristoylation on its own is not sufficient to anchor the protein to the membrane 8. Besides PKC, the MARCKS ED is also a target for Ca2+/ CaM (calmodulin). Activation by CaM initiates another cycle of reversible membrane binding of MARCKS, since CaM will release MARCKS once the intracellular Ca2+ concentration has returned to normal level. If CaM and PKC are both active, they can compete for their common substrate MARCKS. MARCKS can therefore mediate crosstalk between the PKC and CaM signal transduction pathways. If PKC is activated first, MARCKS will become phosphorylated and thus will be removed from the pool of potential CaM-binding proteins. Subsequent activation of CaM would then lead to a stronger activation of CaM substrates such as the important signalling molecules NO (nitric oxide) synthase, myosin light chain kinase (MLCK), CaM kinase II and the phosphatase calcineurin, since phospho-MARCKS cannot compete with them for CaM. In contrast, if CaM is activated first and forms a complex with MARCKS, subsequent activation of PKC will lead to a more pronounced phosphorylation of PKC substrates other than MARCKS.
Functions
Actin cross-link- It has been shown that MARCKS can bind to and cross-link actin in vitro, and that both PKC and CaM inhibit this effect. Thus it was proposed that MARCKS integrates signals from the two pathways into the control of the downstream target and effector molecule, actin. Activation of either of these two pathways would lead to a local release of actin by MARCKS, and a local softening of the actin cytoskeleton with increased plasticity 8.
MARCKS during embryogenesis- Both MARCKS and MRP mRNAs are expressed in the brain and spinal cord from early stages of development. Furthermore, MARCKS proteins are required for embryogenesis, as revealed by several gene knock-out studies 9.
Regulation of actin via PIP2 (phosphatidylinositol-4, 5-bisphosphate) - MARCKS and the MARCKS ED inhibit PLC-induced PIP2 hydrolysis in hosphatidylcholine/phosphatidylserine/PIP2 vesicles. MARCKS regulate the local availability of PIP2.
References
1. Wu WC, Walaas SI, Nairn AC, Greengard P (1982). Calcium/phospholipid regulates phosphorylation of a Mr `` 87k '' substrate protein in brain synaptosomes. PNAS., 79: 5249-5253.
2. Aderem A (1992). The MARCKS brothers: a family of protein kinase C substrates. Cell., 71:713-716.
3. Blackshear PJ (1993). The MARCKS family of cellular protein kinase C substrates. J. Biol. Chem., 268:1501-1504.
4. Bhatnagar RS, Gordon JI (1997). Understanding covalent modifications of proteins by lipids: where cell biology and biophysics mingle. Trends Cell Biol., 7:14-21.
5. Murray D, Ben-Tal N, Honig B, McLaughlin S (1997). Electrostatic interaction of myristoylated proteins with membranes: simple physics, complicated biology. Structure., 5:985-989.
6. Schleiff E, Schmitz A, McIlhinney AJ, Manenti S, Verge' res G (1996). Myristoylation does not modulate the properties of MARCKS-related protein (MRP) in solution. J. Biol. Chem., 271: 26794-26802.
7. Matsubara M, Yamauchi E, Hayashi N, Taniguchi H (1998). MARCKS, a major protein kinase C substrate, assumes non-helical conformations both in solution and in complex with Ca2+-calmodulin. FEBS Lett., 421:203-207.
8. Arbuzova A, Schmitz AP, Vergea-res G (2002). Cross-talk unfolded: MARCKS proteins. Biochem J., 362:1-12.
9. Stumpo DJ, Eddy JRL, Haley LL, Sait S, Shows TB, Lai WS, Young, WS, Speer MC, Dehejia A, Polymeropoulos M, Blackshear PJ (1998). Promoter sequence, expression, and fine chromosomal mapping of the human gene (MLP) encoding the MARCKS-like protein: identification of neighboring and linked polymorphic loci for MLP and MACS and use in the evaluation of human neural tube defects. Genomics., 49:253-264.