Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi

Mrck

Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_101741

Synonyms

Historical Background

MRCK stands for Myotonic dystrophy-Related Cdc42-binding Kinase. The protein earned this name because it binds to Cdc42 with a significant homology to the DMPK (Dystrophia Myotonica Protein Kinase) kinase domain. Prior to the identification of MRCK, many of the RHO-family GTPase effectors homologous to MRCK were discovered. After the identification of the RHO-family GTPases, Rho, Rac1, and Cdc42 (Manser et al. 1994), each of which plays distinctive roles in cytoskeletal reorganization associated with growth and differentiation, the search began for the interacting proteins and potential targets of the p21 (Cdc42/Rac) Rho family that might convey signals downstream to regulate these numerous biological processes. Initially, MRCK was found to interact with Rho for the regulation of stress fibers, focal contact in cells, and to help control cell shape in response to external stimuli (such as lysophosphatidic acid) that are involved in potential changes to the actin cytoskeleton.

The multiple names associated with MRCK, such as PK428 and DMPK-like, are due to the multiple investigators who independently worked on the discovery of the protein. For example, using a PK428 antibody, multiple isoforms of PK428 were immunoprecipitated, cloned, and isolated, including the full-length cDNA clone, a ∼200 kDa protein. PK428 was found to share 65% amino acid identity and a high homology kinase region with DMPK and was shown to interact with the GTP-binding protein Rho, suggesting that there are larger members of this protein kinase family (Leung et al. 1996). Based on its sequence similarity to DMPK, PK428 was proposed to have similar functions (Zhao et al. 1997). Similarities included an overexpression-induced skeletal muscle phenotype, binding to a proline-rich segment (suggesting hematopoietic signaling function), mutations resulting in abnormal cell growth and changes in morphology (supporting a possible role for this protein kinase in cytoskeletal morphology and signal transduction to the nucleus), and an involvement in signaling integrating extracellular events to the cytoskeleton and the nucleus.

Isoforms and Domain Structures

MRCK is a member of the serine/threonine protein kinase family, which includes kinases such as DMPK, Rho-kinase (ROCK), and citron rho-interacting kinase (CRIK), and is conserved in chimpanzee, Rhesus monkey, dog, mouse, chicken, zebrafish, fruitfly, mosquito, C. elegans, and frog. There are three isoforms of MRCK, sharing amino acid sequence and functional similarities, and are products of different genes. As highlighted in Fig. 1, MRCKα is 1719 amino acids in length and encoded by the CDC42BPA gene (Zhao et al. 1997; Tan et al. 2001). This isoform is localized to the same region of chromosome 1 as the gene associated with Rippling muscle disease. MRCKβ is 1711 amino acids in length and encoded by CDC42BPB (Moncrieff et al. 1999). MRCKγ is 1551 amino acids in length and encoded by CDC42BPG (Tan et al. 2003; Ng et al. 2004). MRCKβ and MRCKα are highly homologous, sharing 61% amino acid identity, while the less closely related MRCKγ shares 44% identity with MRCKβ. Homology between the kinase domains reveals an 85% amino acid identity between MRCKα and MRCKβ, while MRCKγ is the nearest homologue with 72% identity relative to MRCKβ.
Mrck, Fig. 1

Schematic structures of MRCK isoforms. Kinase, catalytic kinase domain; CC, coiled-coil; C1, conserved region 1; PH, pleckstrin homology domain; CH, citron homology domain; CRIB, CDC42/Rac-Interactivebinding motif

MRCK contains multiple functional domains and is composed of an N-terminal kinase domain, a coiled-coil structure, and other functional motifs at the C-terminus (Tan et al. 2001). The catalytic kinase domains of MRCK share a high primary amino acid and structural homology with ROCK and DMPK. Hydrophobicity analysis showed MRCK contains a helical region following the kinase domain, as well as a hydrophobic domain. Many of the Rho GTPase effector proteins, such as ROCK, citron, IQGAP, and Dia, contain coiled-coil (CC) regions, which have been shown to facilitate oligomerization (Bishop and Hall 2000). Oligomerization and self-assembly ensures efficient autophosphorylation by increasing the local concentration of catalytic kinase domains. The CC domain of MRCK also shares some homology with the nonmuscle myosin heavy chain C-terminal CC region, which has been shown to entwine to form an extended parallel CC for self-assembly. In the inactive state, MRCK is usually kept in a closed confirmation, held by the stable interaction between the kinase domain and the negative autoregulatory CC domain (Tan et al. 2001). The CC2/3 region of MRCK was found to act as a negative autoregulatory domain for kinase activity by forming a stable complex with the kinase domain and preventing intermolecular N-terminus-mediated kinase dimerization/activation from taking place. This dimerization and subsequent transautophosphorylation (where one subunit of the dimer phosphorylates the other) of the kinase domain are required for MRCK kinase activity.

In addition to their well-conserved kinase domains, all three isoforms of MRCK proteins have protein kinase C conserved region 1 (C1) domains, Pleckstrin Homology (PH)-like domains, and CRIB domains. The C1 domain is a cysteine-rich domain shown to bind to phorbol esters with nM affinities. This phorbol ester binding to the C1 domains may promote MRCK activation (Tan et al. 2001) and/or may contribute to its membrane translocation (Choi et al. 2008). The PH domain is thought to aid in targeting proteins to appropriate subcellular localizations through binding to lipid or protein partners. Adjacent to the PH domain, all three isoforms of MRCK proteins contain a citron homology (CH) domain. The CH domain may mediate protein-protein interactions that specify protein localization or substrate docking. The PH-CH domain tandem is largely responsible for the closed conformation of MRCKβ (Huo et al. 2011). The CRIB domain is a Cdc42/Rac-Interactive Binding motif and has been referred to as PBD (p21 binding domain) due to the original naming of Cdc42. CRIB domain binding induces conformational changes to the adjacent PH-CH domain tandem, thereby exposing MRCKβ zinc binding domain (ZBD) for binding to the tight junction protein ZO-1. Based on MRCKs Rac interactive binding domain and strong binding to Cdc42, MRCK functions as a downstream effector of both Cdc42 and Rac (Fig. 2). MRCK co-localization with Cdc42 induces peripheral actin formation, promotes cytoskeletal reorganization, and stimulates formation of dynamic peripheral structures including microspikes and filopodia (Zhao et al. 1997; Leung et al. 1998; Bishop and Hall 2000). The array and arrangement of C1-PH-CH-CRIB domains in MRCK carboxyl-termini suggests that this region may act in concert to integrate numerous membrane localization signals, specify regions in the membrane with particular lipid/protein composition, and to mediate membrane translocation in response to activation of signaling pathways.
Mrck, Fig. 2

CDC42-MRCK signaling pathway

Role in Cytoskeletal Reorganization

MRCK Contributes to Myosin Light Chain Phosphorylation

There are two major signaling pathways that lead to myosin light chain (MLC) phosphorylation, either Ca2+ mobilization to activate calmodulin-dependent MLC kinases or Rho GTPase-dependent kinase activation, including MRCK, which leads to the regulation of cytoskeletal dynamics and ultimately cell shape. MRCK contributes to MLC phosphorylation to promote cytoskeletal reorganization by binding to Cdc42 (Leung et al. 1996). As shown in Fig. 2, the MRCK kinase domains are able to phosphorylate many substrates, such as MLC, LIMK, and myosin light chain phosphatase (MLCP; Leung et al. 1998). Other kinases also contribute to MLC phosphorylation, including PAK, citron, and ROCK, but their contributions to MLC phosphorylation and biological functions often appear to be distinct and nonoverlapping, which may be due to different sites of activation and/or recruitment. Although MRCK and ROCK share similar substrates, differences in their signaling pathway activation and in their subcellular localization, in basal and/or stimulated states, result in distinct responses. Specifically, MRCK preferentially catalyzes the phosphorylation of Ser19 of MLC, either alone or in combination with Thr18 phosphorylation, which is crucial for activating actin-myosin contractility (Tan et al. 2001). ROCK and MRCK cooperate in regulating MLC2 phosphorylation through MYPT1 phosphorylation. This event regulates contractility by influencing the ATPase activity of the myosin heavy chain (MHC) head groups and leads to actin reorganization.

MRCK Contributes to Cell Contractility and Migration

MRCK has been found to be localized at the periphery of cells, particularly at the leading edge and cell-cell junctions (Leung et al. 1998). Cells form many different shapes based on their function and location in the body, and Rho proteins, such as MRCK, help cells regulate these changes throughout their life-cycle. The MRCK kinases are regulated by Cdc42, which is required for cell polarity, mitosis, and directional migration, and involved in the membrane protrusions of motile cells. Phorbol esters are able to bind to the C1 domain of MRCK and contribute to membrane translocation (Choi et al. 2008).

MRCK is a widely expressed member of the DMPK family that plays key roles in local F-actin organization through a number of kinase and nonkinase effector proteins (Fig. 2). MRCK as well as other Cdc42 effectors act on actin filaments and affect filopodia to propel cells or growth cones across surfaces. Microfilaments generate force when the growing end of the filament pushes against the cell membrane and act as tracks for the movement of  myosin molecules that attach and “walk” along them. Myosin motoring along F-actin filaments generates contractile forces in actomyosin fibers, both in muscle and most nonmuscle cell types. Actin structures are controlled by the Rho family of small GTP-binding proteins, and MRCK links to the actomyosin complex (“stress fibers”) and phosphorylates their light chains, causing contraction of cytoskeletal structures and generating the mechanical force required for cell motility and invasion (Wilkinson et al. 2005; Kale et al. 2014). Actomyosin retrograde flow underlies the myosin contraction essential for elongated morphology, cell motility, and invasion, which is generated by Cdc42-MRCK signaling (Wilkinson et al. 2005).

MRCK also mediates cell-cell adhesion by physically interacting with ZO-1. Co-localization of ZO-1 and MRCK requires the binding of Cdc42 to the kinase and is required for cell migration, as the complex targets the leading edge of cells (Wilkinson et al. 2005; Heikkila et al. 2011; Huo et al. 2011). Based on these interactions and subsequent functions, MRCK can be critical for cancer cell migration, neurite outgrowth, and tissue remodeling during development.

Overexpression in Cancer

MRCK is a key regulator of the actin cytoskeleton and together with multiple target proteins safeguards the tight regulation of normal cell growth and differentiation (Lowe et al. 2012). Elevated MRCK expression levels have been found in various cancers. In the event of genomic alterations or carcinogenesis, cells become predisposed to rapid and uncontrollable growth as demonstrated by the elevated levels of LIMK kinase expressed in prostate cancer and activated by MRCKα. Therefore, inhibitors of MRCK are thought to restore normal cell proliferation and provide a key solution to cancer treatment (Lowe et al. 2012).

MRCK inhibition reduces tumor cell motility and invasion (Unbekandt et al. 2014) by decreasing phosphorylation of MLC (activated form). Inhibition of MRCK has emerged as a potential solution to restoring the tight regulation of normal cellular growth, the loss of which leads to cancer cell formation (Lowe et al. 2012). Elevated levels of ROCK/MRCK substrate proteins MYPT1 and MLC phosphorylation were detected in tumor tissues. Findings indicate therapeutic possibilities in cancers based on interfering with cancer cell migrations by targeting MRCK (Wilkinson et al. 2005). Cycloartane-3,24,25-triol was identified as a potential MRCKα inhibitor due to its ability to compete with an immobilized ligand for binding to ATP-binding sites (Lowe et al. 2012). Chelerythrine was reported to inhibit MRCKα through a non-ATP-competitive mechanism, but the site of ligand binding has not been determined. Poor selectivity makes chelerythrine difficult to use for cell-based experiments to evaluate MRCK function. Inhibition of both MRCK and ROCK inhibits elongated and rounded forms of tumor cell movement and is necessary for full inhibition of specific responses, such as breast cancer cell invasion (Wilkinson et al. 2005). Based on the overlap of substrates as well as their conserved kinase domains, a dual MRCK/ROCK inhibitor may prove therapeutically valuable.

Summary

MRCK is a member of the Rho GTPase family, which operates as molecular switches that are critical regulators of signal transduction pathways in eukaryotic cells. This serine-threonine protein kinase has a high sequence similarity to both DMPK and ROCK. MRCK plays a role in MLC phosphorylation and has been shown to influence cell shape and motility. MRCK has also been shown to be a key regulator of tumor cell migration and invasion. However, our knowledge of the role of MRCK on cancer is limited. Based on our understanding of its normal signaling pathway, its influence on stress fiber formation and contraction, and role in cancer cell migration, MRCK has been acknowledged as a potentially effective therapeutic target.

See Also

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© Springer International Publishing AG 2018

Authors and Affiliations

  1. 1.Penn State University College of MedicineHersheyUSA