Myristoylated Alanine-rich C-Kinase Substrate (MARCKS): Its Role in Cell Adhesion and Cell Spreading

Cells are grouped together in order to form into three-dimensional tissues of animals. They are joined together with the purpose of establishing the tissues’ design or architecture. While tissues have distinct properties and organization, their grouping is based on a vital element- cell adhesion. There is a wide array of methods used by the body for cell adhesion. Regarded as a mechanism, cell adhesion aids in translating “genetic information into the complex three-dimensional patterns of cells in tissues” (Gumbinger, 1996).
Recently, scientists began studying the role of Myristoylated Alanine-rich C-Kinase Substrate (MARCKS), a major substrate of protein kinase C (PKc) in cell adhesion and cell spreading. The aim of this paper is to elucidate how MARCKS is involved in cell adhesion and cell spreading. To understand this role, the first section of this paper will briefly consider how cell adhesion occurs in a fully formed tissue. Widely accepted mechanisms of cell adhesion will be briefly cited in this section. The second section will be devoted on the vital function that MARCKS play in cell adhesion and cell spreading. This section will also show the function of MARCKS in development of tissues.

Cell Adhesion in a fully formed tissue
How cells adhere to each other is brought about by complexes of proteins. These complex proteins are subdivided into cell adhesion molecules and cell adhesion receptors; the peripheral membrane proteins and the extracellular matrix proteins. The contacts between cells and between cells and matrix are brought about by three factors: adhesive systems, the actin cytoskeleton and the force generated in the regions of the cell (Gumbinger, 1996).
The following group of proteins are accepted to play important roles in cell adhesion (Petruzzelli, L. et. al., 1999; Yamada,K. 2002; Gumbinger, 1996): cadherin family; Integrin family; immuglobulin superfamily and selectin superfamily. The succeeding paragraph will briefly state the characteristics or function of each group of proteins in bullet points.
Cadherin Family

• Transmembrane proteins that have five motifs bridged by calcium
• Removal of calcium makes allows it to lose its adhesive property and makes it vulnerable to proteases

Integrin Family

• Heterodimers that have both alpha and beta subunits
• The linkage between heterodimers are noncovalent
• Has low affinity with their ligands
• Allows cell adhesion to their matrix

Immunoglobulin family

• Most are also transmembrane proteins
• Structurally most diverse
• Have domains that serve as binding sites of antigens
• Send out signals during an immune response

Selectin superfamily

• Transmembrane proteins
• Has a N-terminal lectin domain
• Has an EGF-type domain
• Expressed on the surface of leukocytes, endothelial cells

Role of MARCKS in Cell Adhesion and Cell Spreading
Myristoylated Alanine-rich C-Kinase Substrate (MARCKS) is the major substrate of protein kinase C (PKC) in many cells and tissues. Their binding to actin filaments, calmodulin and acidic membrane phospholipids are all controlled by protein phosphorylation (Yamauchi, et. al., 1998).It has also been suggested that aside from cell adhesion, MARCKS is also responsible for secretion, motility and mitogenesis of tissues. These events are thought to be regulated by the actin-cytoskeletal structure.
MARCKS also play a role in fibrogenesis in liver cells. A study linking MARCKS to platelet-derived growth factor (PDGF) signaling pathway was conducted using the human hepatic stellate cells (hHSC). The group of researchers conducting the study concluded that MARKCS has a vital function in fibrogenesis (growth of fibers) in the liver. In addition to secretion and motility, MARCKS was found out to play an important role in the chemotaxis of activated hHSC (Rombouts, et.al, 2008).
A study done by Myat and his colleagues (1997) elucidated the integral role of MARCKS in cell spreading and membrane ruffling. The research team acknowledged that Protein Kinase C (PKC) is important in stimulating membrane ruffling and subsequent adhesion. While the mechanism on how this happens is not clear, the researchers investigated how MARCKS is involved in the process. Their results provided direct evidence that MARCKS and PKC control “actin-dependent membrane ruffling and cell adhesion, perhaps via a PIP-2 (phophatidylinositol 4,5 biphosphate) dependent mechanism”. (Myat, et.al. 1997)
Meanwhile, an important step involved in myogenesis is myoblast migration. Researchers demonstrated that calcium activated neutral proteases or Calpains are vital in cell spreading and motility. During myoblast migration, they found out that there is a direct relationship between the level of Calpain and myoblast migration. Once the level of Calpain is reduced, myoblast migration is also reduced. However, the level of MARCKS, which is present at adhesion sites, accumulates once Calpin is reduced. At the conclusion of their study, they found out that “normal myoblast migration is dependent on MARCKS phosphorylation and localization”. (Dedieu, S. & et. al, 2003)
During tissue morphogenesis, the regulation of cytoskeleton plays a critical role in the normal functioning of the cell (Disatnik, M. et. al, 2004). Integrins regulate the cytoskeleton during the interaction between the cell and the matrix. However, little is known about the signaling cascades that govern the process. Disatnik and her colleagues showed that MARCKS play a vital role during muscle cell spreading. MARCKS is regulated by 5ß1 integrin through activation of PKC and in the process; MARCKS regulate the formation of actin stress fiber during spreading of the muscle cells. They reported that the translocation of MARCKS is bidirectional (i.e. tranlocation first occurs from the membrane then to the cytosol and back again to the membrane). According to this group, “this type of bi-directional translocation of an actin regulatory protein highlights the importance of the temporal control of actin dynamics during integrin-mediated cell adhesion and spreading”. (Distanik, M. et. al., 2004)
Meanwhile, PKC through its physiological substrate, MARCKS modulates the growth of melanocytes in a laboratory culture. Researchers (Brookes, et. al, 1996) found out that aside from promoting cell adhesion and cell spreading, MARCKS appear to play a role as a growth suppressor in the differentiation of cells that have a melanocyte origin. Perhaps, MARCKS have a vital role in suppressing the growth of carcinomas.
Meanwhile, MARCKS is also ‘involved in synaptic plasticity’ (Higo, N., et.al., 2002). A MARCKS related protein (MRP) also participates in organization of actin cytoskeleton, focal contact distribution and cell morphology when its expression is controlled by Integrins. Both MARCKS and MRP have an important role in cell spreading (Bout, I., et. al., 2007). While MARCKS is also implicated in the regulation of “cell spreading, stress fiber formation, and focal adhesion assembly in nonmuscle cells” (Heidkamp, M., et. al., 2007), its role in growth of cardiomyocyte has not been fully explored until Heidkamp (2007) and her colleagues studied this phenomenon. While an earlier study did explore the expression and regulation of MARCKS in a developing rat heart (McGill, 1997), the study of Heidkamps group further elucidated MARCKS role in the myocytes of rats. In their study, they found out that MARCKS is also expressed in both neonatal and adult rat ventricular myocytes in the laboratory. This suggests that MARCKS is also involved in cell adhesion in heart muscle cells.
MARCKS, along with GAP43, CAP23, are substrates of PKC that play a role in actin cytoskeleton. These three proteins are important in both morphogenic processes and cell motility. They are also present at plasmalemmal raft and through a common mechanism, regulate both cell cortex and actin cytoskeleton (Laux, T., et. al., 2000).
Aside from the regulating morphogenesis in liver tissues and heart muscle tissues, MARCKS has also a role “in the integrin-dependent signal transduction pathways in macrophages” (Li,J., et.al., 1996). MARCKS was also discovered to have a role in sequestering phophatidylinositol 4,5 biphosphate in lipid bilayers (PIP(2)). PIP(2) is a signal molecule present in the cell membrane and is implicated to regulate a number of ion channels and transporters (Rauch, M. et. al, 2002).

SUMMARY
MARCKS play a role in cell adhesion and cell spreading in muscle cells by binding to actin filaments, calmodulin and acidimmembrane phospholipids. In muscle cells, MARCKS control muscle spreading by regulating the formation of actin stress fibers. In non-muscle cells, MARCKS help in formation of stress fibers, focal adhesion assembly and regulation of cell spreading. MARCKS is also expressed in both neonatal and adult ventricular myocytes of laboratory rats and play an important role in both cell adhesion and spreading. It also has a role in liver fibrogenesis, cell spreading and ruffling and is involved in synaptic plasticity. In morphogenesis, MARCKS is vital in the normal myoblast migration. It also regulates cell motility and has a role in the signal transduction pathways of macrophages and regulates a number of ion channels and transports as well. Interestingly, MARCKS also modulates the growth of melanocytes and appear to have a role in suppressing the growth of carcinomas.
Playing an important part in cell adhesion and cell spreading, MARCKS, as an important substrate of Protein Kinase C (PKC) is indispensable in the formation of the 3-dimensional structure and architecture of tissues. It allows the proper migration and adhesion of cells and supports the continuity and integrity of tissues.