Contraction of vascular smooth muscle cells occurs when the free Ca²⁺ levels in the cytosol increase. This happens through calcium influx from the interstitium and calcium discharge from the intracellular reservoirs. A range of different triggers (e.g., electrical signals from nerves, different ligands from the blood, or changes of intraluminal pressure) can increase calcium levels.^[2,12,13] The Ca²⁺ and calmodulin (a key Ca²⁺-binding protein and intracellular signal transducer) bind together to create a calcium-calmodulin complex that subsequently activates the myosin light chain kinase (MLCK).^[14] Afterwards MLCK phosphorylates the Ser-19 residue on the 20 kDa myosin regulatory light chain (MLC₂₀).^[13,14] This interplay initiates the actomyosin cross-bridge cycle by increasing the actomyosin ATPase activity. This process activates the contractile machinery and the vascular smooth muscle cells contract^[15,16](Fig. 1).

Other mechanisms not involving [Ca²⁺]_i contribute to the regulation of vascular tone. For instance, protein kinase C (PKC) and Rho-associated kinase (ROCK) inhibit the myosin light-chain phosphatase (MLCP).^[20] This interaction triggers the phosphorylation in MLC20, causing vascular contraction without a significant change in [Ca²⁺]_i, a process known as Ca²⁺-sensitization.^[13] Other intracellular second messenger molecules, such as cAMP and cGMP, can cause relaxation by boosting the activity of MLCP, which leads to the opposite effect of MLC₂₀ dephosphorylation. The latter mechanism is defined as desensitization.^[13]

MLCP is a trimeric enzyme, consisting of a catalytic subunit (PP1cδ), a regulatory subunit myosin phosphatase targeting subunit (MYPT1), and a small 20- to 21-kDa subunit whose function is not completely understood.^[17] It is widely expressed in various cell types and can catalyze the dephosphorylation of different intracellular protein substrates.^[18] There are three known mechanisms for regulation of MLCP-holoenzyme activity (Fig. 1), i.e., dissociation from the heterotrimeric structure, inhibition by intracellular regulatory proteins such as PKC-potentiated inhibitory protein of 17 kDa (CPI-17), or regulation of the enzyme by the targeting subunit of MLCP, MYPT1.^[17]

Among the aforementioned mechanisms regulating MLCP activity, the phosphorylation of MYPT1 seems to be the most important one. The activity of MLCP is shown to relate to the degree of MYPT1-threonine phosphorylation at position 696 (MYPT1-T696)^[19], as the ablation of this phosphorylation site is lethal in homozygous or heterozygous form.^[20,21]MYPT1-T696 is phosphorylated by many protein kinases, such as ROCK, PKC, ILK, ZIPK, and possibly RSK2, as these phosphorylations are attributed to inhibition of MLCP and Ca²⁺-sensitization.^[18]ROCK can also phosphorylate MYPT1 at T853, which results in reduced phosphatase activity.^[22] Other enzymes, such as PKG and PKA, phosphorylate phosphoserine MYPT1 sites, S668 and S695, to desensitize vascular smooth muscle cells to Ca²⁺, which in turn leads to relaxation^[15,23,24](Fig. 1). S695 has no direct effect on MLCP activity, but its high pre-phosphorylation interferes with the ability of ROCK or other kinases to phosphorylate MYPT1-T696 and inhibits MLCP.^[24]MYPT1 phosphorylation seems to be involved in the control of cell proliferation, as the degree of phosphorylation at Ser473 rises during mitosis.^[25]

Cloning and sequencing from various tissues and species have revealed the existence of various MYPT1 isoforms generated by alternative splicing. The splicing of Exon 24 of MYPT1 mRNA (MYPT1E24) in smooth muscle tissues has been well studied^[26-28], showing that including or skipping this exon creates MYPT1 isoforms with different leucine zipper motifs (LZ), which changes the reactivity of vascular smooth muscle to agonists or protein-kinase-G.^[15,29] It has been demonstrated that in large arteries and veins where tonic smooth muscle is abundant, the smooth muscle cells express predominantly the E24-skipped variant for the C-terminal LZ motif (E24-/LZ+).^[26] The MYPT1E24-/LZ+ version is mostly found early in development in tissues with a phasic contractile phenotype, such as the portal vein and the intestines.^{[26,28,30,31]} Researchers believe that the shift in MYPT1 isoforms contributes to the diversity of smooth muscle contractile phenotypes as smooth muscles develop.^[32] It has been observed that smooth muscle cells in cultures express only the slow isoform, suggesting that the tonic phenotype is the default isoform.^[32] In simpler terms, large blood vessels that are likely to develop the tonic phenotype express the slow program gene and the MYPT1E24−/LZ+ isoform early in their development, and this pattern continues into adulthood. In contrast, the MYPT1E24+/LZ- isoform is mainly found in coronary, mesenteric, or femoral arteries.^[27,33,34] The large blood vessels in the brain represent an intermediate form expressing to a similar extent the MYPT1, with or without E24^[15], which may be linked to their functional role in rapid flow redistribution matched to the metabolic demand and activity of brain tissue. The MYPT1E24− isoform is present in the arteries from phasic phenotypes, such as the femoral artery in senescent mice^[34], which suggests that MYPT1 isoforms could influence the blood pressure adaptation in advanced age. Even though these findings show that MYPT1 isoforms play a role in vascular contractility, further validation in vivo is required to confirm this.

Inflammatory bowel diseases (like Crohn’s disease and ulcerative colitis) are chronic, relapsing conditions that cause ongoing inflammation in the digestive tract and disrupt normal intestinal motility.^[39] A key mediator of the inflammatory response is interleukin-1β, which activates various immune cells but can also influence intestinal motility.^[36] Ohama et al. recently demonstrated that long-term treatment of ileal smooth muscle cells from Wistar rats with this mediator lowers the levels of phosphorylated MYPT1, which can result in less force being produced.^[36]MYPT1 protein is also reduced in Hirschsprung’s disease. This disease, also known as congenital aganglionic megacolon, results from a lack of both the Meissner submucosal and the Auerbach myenteric plexus^[40], leading to functional obstruction, failure to pass meconium, and increasing the risk of bacterial infections.^[41] Furthermore, others offered an explanation for the mechanism of colonic obstruction by treating proximal and distal colonic smooth muscle cells from mice with lipopolysaccharide. The results showed substantial MYPT1 protein degradation caused by SIAH1/2 E3 ligases through the ubiquitin proteasomal pathway, which affects how the colon contracts and causes obstruction.^[42]MYPT1 protein levels were significantly lower in the smooth muscle of the stomach in people with diabetes, indicating that the modulation by Ca²⁺-sensitization can play a crucial role in diabetes-induced gastroparesis.^[43] Moreover, in a rat diabetic model, the levels of arterial MYPT-T696/853 were significantly higher^[44], indicating that smooth muscle hypercontractility is linked to a major reduction in MLCP activity.

MYPT1 can affect contractility and microtubule acetylation to regulate matrix assembly and integrin adhesions.^[45] In patents, studies have recently demonstrated that MYPT1 expression levels were reduced in gastric cancer and that patients with increased MYPT1 levels had a longer survival rate.^[46] The same group has demonstrated in cell cultures that overexpression of MYPT1 inhibits gastric cancer cell division, migration, and invasion through inhibition of RhoA phosphorylation^[46], making MYPT1 a good candidate for a gastric cancer biomarker and possibly a target therapy. Another intriguing role of MYPT1 is in ovarian cancer as a tumor suppressor gene. The Hippo pathway, which regulates the expression of genes influenced by Yes-associated protein (YAP), is dependent on MYPT1. If MYPT1 is downregulated, this can lead to a lower response to therapy with platinum-based drugs such as cisplatin, carboplatin, and oxaliplatin, which are commonly used as anti-cancer drugs.^[47,48] Last but not least, MYPT1 has been shown to slow down the progression of non-small cell lung carcinomas by inhibiting β-catenin signaling.^[49]

When it comes to expression of MYPT1 in the pulmonary arteries, it has been found that both MLCK and MLCP decrease with the progression of age, with the sharpest decrease occurring after birth. This decrease might correlate to the high resistance in these arteries during the prenatal period and the quick relaxation occurring in the postnatal period.^[38] Another study found that preserving the expression of MYPT1 and MYPT1 (LZ+) in porcine pulmonary arteries helps maintain their response to cGMP and prevents the development of NO tolerance.^[50]

A relatively recent study has shown the role of MYPT1 in gallbladder contractility and the hepatic damage that may occur if it is downregulated.^[37] This study discovered that reducing MLCP activity inhibits Cell Counting Kit-8 responsiveness, resulting in dysfunctional bile collection and hepatic fibrosis.^[37]