Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi

MYLK (Myosin Light Chain Kinase)

Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_248

Synonyms

Historical Background

The gene for myosin light chain kinase encodes three proteins: MLCK210, MLCK108, and telokin/kinase-related protein (KRP). The first protein discovered was MLCK108 (Mr = 110–140 kDa) as a major cytoplasmic component of smooth muscle and responsible for smooth muscle contractility through the phosphorylation of the regulatory light chain of myosin (Lukas et al. 1998; Kamm and Stull 2001). MLCK210 (Mr = 210–220 kDa) has an amino-terminal extension containing additional protein-binding elements (Fig. 1). It was discovered ∼15 years later before the gene for MYLK was characterized from chicken (Birukov et al. 1998) and humans (Shen et al. 2012). Both MLCKs are Ca2+-calmodulin-dependent enzymes. Telokin/KRP is an independently expressed nonkinase gene product containing the C-terminus of MLCK, and functions as a myosin-binding and filament-stabilizing protein (Shirinsky et al. 1993). Telokin/KRP is primarily expressed in smooth muscle and modulates contractility by regulating the access of protein kinases to myosin light chains (Shcherbakova et al. 2010). It has also been implicated in activation of the myosin light chain phosphatase MLCP through the interaction with its regulatory subunit MYPT1 and relief of phosphorylation-induced inhibition of MLCP activity (Khromov et al. 2012).
MYLK (Myosin Light Chain Kinase), Fig. 1

Domain structure of myosin light chain kinase gene products. This image has a schematic depicting each protein and its domain structure

MLCK-Domain Structure and Human Genetics

As shown in Fig. 1, MLCK is a multidomain protein. MLCK108 has amino-terminal actin-binding motifs, a protein kinase catalytic domain, calmodulin regulatory domain, and the myosin-binding (telokin/KRP) domain. Within the amino-terminal domains are IgG C2-like domain motifs and fibronectin-like motif that may be mediators of other protein–protein interactions within the cell. Similarly, the amino-terminal extension found in the MLCK210 isoform has six additional IgG domains and actin-binding motifs (Kamm and Stull 2001). The additional actin-binding motifs in MLCK210 allow stronger interactions with the actin cytoskeleton (Kamm and Stull 2001), while the expanded IgG domain motifs may be associated with binding to other proteins such as tubulin (Kudryashov et al. 2004), macrophage migration inhibition factor (MIF) (Wadgaonkar et al. 2005), cortactin (Shen et al. 2012), and supervillin (Takizawa et al. 2007).

Single nucleotide polymorphisms (SNPs) in the mylk1 gene (Human chromosome 3q21) have been associated with two conditions: severe inflammatory lung disease, including asthma, and sepsis (Shen et al. 2012). It is interesting that African-derived and Caucasian populations differ in the frequency of various polymorphisms, which may make them risk factors in one population, but not the other. For example, a haplotype-containing SNP rs9361070, which is within the promoter region of MLCK108, was negatively associated with asthma (decreased risk) in both American and Caribbean families. However, the same haplotype conferred risk for severe sepsis (Shen et al. 2012). Microarray studies of mRNA from blood cells in the various patient population indicated that there was a decrease in MLCK expression associated with the minor allele of SNP rs9361070. This may explain the reduced risk of asthma, because MLCK expression is upregulated in asthmatic airway tissue (Stephens et al. 2007). African Americans are also at higher risk for glaucoma and there is an increased expression of MLCK108 in the optic nerve head astrocytes of African Americans compared to Caucasian populations, although specific polymorphisms have not been identified with disease. On the other hand, MLCK210 is upregulated in glaucomatous astrocytes of both populations (Lukas et al. 2008). Finally, upregulation of MLCK210 is associated with inflammatory bowel disease, but as with glaucoma, no polymorphisms have been associated with disease (Cunningham and Turner 2012).

Expression of MLCK Isoforms

MLCK108 is expressed in most tissues, with lower levels appearing in embryonic cells and precursors. MLCK210 specific expression occurs predominantly in endothelial cells of blood vessels and epithelial cells of the intestine and lung. Primary cells such as those from smooth muscle often exhibit decreased expression of MLCK108 when placed in tissue culture (Herring et al. 2006).

MLCK108, MLCK210, and telokin/KRP expression are independently regulated. Figure 2 illustrates the relationships among the transcripts produced from the mylk1 gene. Several serum response factor (SRF) elements that are often associated with smooth muscle gene expression are located in MLCK108 promoter and the first MLCK108 intron. Promoter analysis indicates that myocardin, an SRF activator, is necessary for high-level expression of MLCK108 and telokin/KRP in smooth muscle. GATA-6, an SRF repressor, may be responsible for the downregulation of MLCK108 and telokin/KRP in nonmuscle tissues (Herring et al. 2006). In addition to SRF, other transacting factors that affect MLCK108 and/or telokin/KRP expression include thyrotroph embryonic factors (TEF), Foxf1, and Hox proteins. These factors bind to AT-rich regions adjacent to the SRF sites (Herring et al. 2006; Khapchaev and Shirinsky 2016). MLCK108 promoter activity is also enhanced by Notch1 complex operating in an autoregulatory mode through the simultaneous induction of HRT2 transcription factor that represses both Notch1 and SRF/myocardin (Basu and Proweller 2016).
MYLK (Myosin Light Chain Kinase), Fig. 2

Promoters within the myosin light chain kinase gene. Upper part of the figure describes where the four promoters are located and shows mRNA for each product of the mylk1 gene. Below the approximate location of transcription factors binding motifs within MLCK108 and MLCK210 promoters are depicted together with the known interactions between these factors and inducing agents. Green color denotes activation and red/brown color denotes repression of transcriptional activity. Additional explanations in the text. Scheme is the courtesy of Dr. Asker Khapchaev (Russian Cardiology Research and Production Complex, Moscow, Russia)

In the case of MLCK210, the promoter is more complex because there are two alternate start sites of transcription each containing its own upstream promoter (Fig. 2). MLCK210 expression in epithelial cell types is controlled by the proximal promoter. Its basal activity is maintained by p53 and Sp-1 transcription factors and is further induced by proinflammatory agents such as tissue necrosis factor alpha ( TNFα), interleukin-1beta (IL-1β), interferon-gamma (INF-γ), and hypoxia (Cunningham and Turner 2012). Proinflammatory agents increase MLCK210 expression through NFκB transcription factor perturbing balance between the repressing NFκB dimers (р50/р50) and activating NFκB heterodimers (p50/p65) in favor of the latter (Al-Sadi et al. 2016).  TNFα, INF-γ, and hypoxia signaling may converge on a hypoxia-induced factor-1alpha (HIF-1α) or its antagonist FIH. Additionally, IL-1β through Erk1/2 and p38 MAP kinase signaling cascades activates Elk-1 and ATF-2 transcription factors that enhance MLCK210 expression (Khapchaev and Shirinsky 2016). Another expression-enhancing module in this promoter consists of a triple binding site for AP-1 transcription factors. The relative importance of NFĸB compared to AP-1 transcriptional activity is dependent on the extent of cell differentiation with NFĸB declining and AP-1 increasing with cell confluence (Cunningham and Turner 2012). In endothelial cells expression of MLCK210 is accomplished from a distal promoter, which is located about 50 kb upstream in mylk1 gene from the proximal promoter. Transcriptional activity from this promoter is induced by Vascular Endothelial Growth Factor (VEGF) and mediated by Sp-1 transcription factor and other currently uncharacterized factors (Shimizu et al. 2015).

The mylk1 gene products are the subject for negative posttranscriptional control by microRNA (miR), a class of small endogenous noncoding RNA that regulate mRNA levels of many proteins. MiR-374a, miR-374b, miR-1290, miR-520c-3p, and miR-155 bind 3′-noncoding region of MLCK210 mRNA in an additive fashion (Adyshev et al. 2013; Weber et al. 2014). Manipulations to reduce the levels of miR-1 result in enhanced expression of MLCK210 in endothelium and of telokin/KRP in cardiomyocytes. Similarly, decreased expression of miR-200c in several cancer cell lines and in breast cancer patients correlates with increased MLCK expression (Khapchaev and Shirinsky 2016). Additionally, two noncoding RNAs, MYLK-AS1 and MYLK-AS2, were identified in the antisense DNA strand of the mylk1 gene. Part of MYLK-AS1 sequence is complementary to the coding sequence of MLCK108 and MLCK210 providing for possible role of MYLK-AS1 in posttranscriptional regulation of MLCKs (Khapchaev and Shirinsky 2016).

MLCK and Signal Transduction

As mentioned earlier, MLCK108 is one of the kinases responsible for Ca2+-stimulated contraction of smooth muscle (Kamm and Stull 2001). Ca2+-calmodulin-dependent activation is further modulated by phosphorylation at serine residues within the calmodulin regulatory region (Fig. 3). This region has two subdomains that function to maintain the kinase in an autoinhibited state that is released when calmodulin binds and activates the enzyme (Lukas et al. 1998). Site-directed mutagenesis studies established the importance of selected charged residues within the autoinhibitory- and calmodulin-binding domains with respect to autoinhibition and calmodulin binding. Calmodulin-dependent protein kinase II, protein kinase C, PAK2, AMP-dependent protein kinase, cyclic nucleotide (cyclic AMP, cyclic GMP)-dependent protein kinases, and MLCK itself are capable of phosphorylating sites within the calmodulin-binding domain (serines 1759/1760 in human MLCK210; Fig. 3) and decrease the Ca2+ sensitivity of MLCK activation. On the other hand, phosphorylation of MLCK by MAP kinase stimulates activity in cultured tumor cells (Khapchaev and Shirinsky 2016). Tyrosine kinase Src phosphorylates Y464 and Y471 in MLCK210 isoform 1 but not in isoform 2 in which these tyrosine residues are spliced out. Although these sites are distal from the calmodulin and kinase catalytic domains, tyrosine phosphorylation slightly increases MLCK210 activity. Tyrosine phosphorylation of MLCK210 in cultured cells has been observed after stimulation with a tyrosine phosphatase inhibitor or constitutively activated epidermal growth factor (EGF) receptor (Kamm and Stull 2001).
MYLK (Myosin Light Chain Kinase), Fig. 3

Calmodulin regulatory domain of MLCK. This domain contains the autoinhibitory and calmodulin recognition region and a listing of protein kinases that modulate Ca2+-calmodulin binding through phosphorylation at the C-terminal end of this region. The legend indicates the protein kinases that phosphorylate one of the two serines within the RLSS sequence

In addition to the identified sites at tyrosine 464/471, and serine 1759/1760, MLCK210 has multiple other posttranslational modifications (PTM) in its primary structure. To date about forty phosphorylated sites and nearly a dozen acetylated sites have been discovered in MLCK210 using mass spectrometry and other approaches (Hornbeck et al. 2015). These PTMs are likely dynamically regulate enzymatic activity, localization, and signal integrating function of this protein (Table 1). For example, N-terminal domain of MLCK210 is differentially phosphorylated and localized in mitotic and interphase cultured cells suggesting that phosphorylation (by the kinase Aurora B) may impact MLCK210 cellular localization (Dulyaninova and Bresnick 2004). Subsequent work demonstrated that phosphorylation of Ser-140/149 in chicken MLCK210 N-terminal construct (homologs of Ser-145/154 in human MLCK210) by PKA and Aurora B in vitro alters the actin filament binding whereas phosphomimicking mutant of MLCK210 N-terminal tail has reduced binding to the cytoskeleton in cultured cells (Vilitkevich et al. 2015). In another study, a lysine acetylase ARD1 targeted Lys-608 in MLCK210 in a Ca2+-CaM-dependent fashion. Acetylation of MLCK210 reduced myosin light chain phosphorylation in cancer cells and inhibited MLCK-dependent invasion and migration (Shen et al. 2012).
MYLK (Myosin Light Chain Kinase), Table 1

MLCK210 phosphorylation in vitro, in living cells, and tissues (Data obtained from http://www.phosphosite.org (Hornbeck et al. 2015))

Phosphorylated residuea

Protein kinase

Functional significance

S-154

PKA, Aurora B

↓ Interaction with actin filaments

Y-471

Src

↑ MLCK210 enzymatic activity and interaction with cortactin

Y-611

c-Abl

Uncertain

S-1208

PAK2

Unknown

S-1388

PKA

Unknown

Y-1449

c-Abl

Unknown

S-1759

PAK2

↓ Interaction with CaM and MLCK enzymatic activity

S-1760

PKA, PKG, CaMKII, PKC, AMPK, MLCK (autophosphorylation)

↓ Interaction with CaM and MLCK enzymatic activity

S-1768

MLCK (autophosphorylation)

Unknown

S-1772

PAK1

Unknown

S-1773

PKA, PKG, ERK2, CaMKII

Possibly ↑ interaction with MYPT1 subunit to overcome phosphorylation-induced inhibition of MLCP enzymatic activity

S-1776

PKA

Unknown

S-1779

ERK2, PKA, PKG

Unknown

aOnly phosphosites identified by site-specific methods such as amino acid sequencing, site-directed mutagenesis, phosphosite-specific antibodies, etc. and confirmed by proteomic discovery-mode mass spectrometry are listed; numbering is for human MLCK210 isoform 1

MLCK210 as a Signal Integrator

The interactions of MLCK210 with various kinases as well as with specific proteins suggest that MLCK210 may coordinate (integrate) signaling from other cellular processes (Kudryashov et al. 2004). This may be particularly important in the cell types that express MLCK210 and contain mostly actin and little myosin, so that MLCK catalytic activity contributes toward changes in cell morphology and/or migration rather than a contractile event as found in smooth muscle. A summary of such signal integrations is illustrated in Fig. 4. Clearly, not all of these interactions happen in every tissue/cell type. However, selected sets of interactions and signaling events that regulate MLCK localization and activity are likely to play a role in specific cellular processes. Interactions between MLCK210, MIF, and actomyosin fibers of the cytoskeleton may be important in endothelial cells involved in an inflammatory response (Wadgaonkar et al. 2005). Likewise, recruitment and activation of Pyk2 tyrosine kinase by MLCK210 is required for full activation of β 2 integrins and neutrophil transmigration through microvascular wall in sepsis-induced lung inflammation model (Xu et al. 2008). Membrane-associated scaffolding protein supervillin also directly interacts with MLCK210 N-terminus as well as with myosin II. Through these interactions, supervillin apparently modulates myosin II activation by MLCK210 and contributes to myosin II assembly during cell spreading (Takizawa et al. 2007). Interaction of MLCK210 with cortactin may exert regulatory action on cortactin-Arp2/3 complex that mediates assembly of the cortical actin network in endothelial cells and controls endothelial barrier (Shen et al. 2012). As demonstrated above, the interactions of MLCK210 with its protein partners may be regulated by specific posttranslational modifications introduced in a vicinity of a protein-binding interface or elsewhere. Currently, the functional significance of the majority of PTM in MLCK210 is not known driving the future research in this important direction.
MYLK (Myosin Light Chain Kinase), Fig. 4

MLCK210 (central molecule in the figure extended from left to right side) contains multiple interaction sites for cytoskeletal and regulatory proteins. Shown in this figure are selected binding partners for MLCK210 that interact with MLCK210 as well as those shared by MLCK108 (Actin, CaM, myosin, etc.). Protein kinases and ARD1 acetylase that modify MLCK210 residues (approximate position is shown by arrows) are listed in red/dark red color. Relative protein sizes are not to scale

MLCK 210 and Barrier Function

Barrier function in lung vascular endothelium is regulated by agonist induced MLCK activation. Further regulation by miRNA (Adyshev et al. 2013), phosphorylation (Cunningham and Turner 2012), and inflammatory cytokines (Shimizu et al. 2015) indicates that barrier dysfunction is mediated by MLCK210. This concept was substantiated by the MLCK210 KO mouse which has much lower susceptibility to lung damage (Khapchaev and Shirinsky 2016). Similarly, epithelial MLCK-210 is implicated in intestinal (Cunningham and Turner 2012) and esophageal (Tan et al. 2014) inflammation leading to inflammatory bowel disease or gastroesophageal reflux disease. Alterations of MLCK210 expression/activity in multiple human diseases indicate that MLCK210 is a potential target for pharmacological and/or genetic intervention.

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Copyright information

© Springer International Publishing AG 2018

Authors and Affiliations

  1. 1.Department of PharmacologyNorthwestern UniversityChicagoUSA
  2. 2.Institute of Experimental CardiologyRussian Cardiology Research and Production Complex, Ministry of Healthcare of Russian FederationMoscowRussia