Myosin-light-chain phosphatase

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Myosin Light-Chain Phosphatase
Complex between PP1 and a portion of MYPT1 structure.png
Structure of complex between PP1 and a portion of MYPT1, generated from 1s70 [1]
Identifiers
EC no. 3.1.3.53
CAS no. 86417-96-1
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Myosin light-chain phosphatase, also called myosin phosphatase (EC 3.1.3.53; systematic name [myosin-light-chain]-phosphate phosphohydrolase), is an enzyme (specifically a serine/threonine-specific protein phosphatase) that dephosphorylates the regulatory light chain of myosin II:

Contents

[myosin light-chain] phosphate + H2O = [myosin light-chain] + phosphate

This dephosphorylation reaction occurs in smooth muscle tissue and initiates the relaxation process of the muscle cells. Thus, myosin phosphatase undoes the muscle contraction process initiated by myosin light-chain kinase. The enzyme is composed of three subunits: the catalytic region (protein phosphatase 1, or PP1), the myosin binding subunit (MYPT1), and a third subunit (M20) of unknown function. The catalytic region uses two manganese ions as catalysts to dephosphorylate the light-chains on myosin, which causes a conformational change in the myosin and relaxes the muscle. The enzyme is highly conserved [1] and is found in all organisms’ smooth muscle tissue. While it is known that myosin phosphatase is regulated by rho-associated protein kinases, there is current debate about whether other molecules, such as arachidonic acid and cAMP, also regulate the enzyme. [2]

Function

Smooth muscle tissue is mostly made of actin and myosin, [3] two proteins that interact together to produce muscle contraction and relaxation. Myosin II, also known as conventional myosin, has two heavy chains that consist of the head and tail domains and four light chains (two per head) that bind to the heavy chains in the “neck” region. When the muscle needs to contract, calcium ions flow into the cytosol from the sarcoplasmic reticulum, where they activate calmodulin, which in turn activates myosin light-chain kinase (MLC kinase). MLC kinase phosphorylates the myosin light chain (MLC20) at the Ser-19 residue. This phosphorylation causes a conformational change in the myosin, activating crossbridge cycling and causing the muscle to contract. Because myosin undergoes a conformational change, the muscle will stay contracted even if calcium and activated MLC kinase concentrations are brought to normal levels. The conformational change must be undone to relax the muscle. [4]

When myosin phosphatase binds to myosin, it removes the phosphate group. Without the group, the myosin reverts to its original conformation, in which it cannot interact with the actin and hold the muscle tense, so the muscle relaxes. The muscle will remain in this relaxed position until myosin is phosphorylated by MLC kinase and undergoes a conformational change.

Structure

A 3D representation of PP1 (shown in red) and a portion of MYPT1 (shown in blue), with the manganese ion catalysts shown in white. The yellow lines mark the grooves that are critical for enzyme binding and catalysis. PP1 and a portion of MYPT1 with the Manganese ion catalysts 3D.png
A 3D representation of PP1 (shown in red) and a portion of MYPT1 (shown in blue), with the manganese ion catalysts shown in white. The yellow lines mark the grooves that are critical for enzyme binding and catalysis.

Myosin phosphatase is made of three subunits. The catalytic subunit, PP1, is one of the more important Ser/Thr phosphatases in eukaryotic cells, as it plays a role in glycogen metabolism, intracellular transport, protein synthesis, and cell division as well as smooth muscle contraction. [5] Because it is so important to basic cellular functions, and because there are far fewer protein phosphatases than kinases in cells, [6] PP1’s structure and function is highly conserved (though the specific isoform used in myosin phosphatase is the δ isoform, PP1δ). [7] PP1 works by using two manganese ions as catalysts for the dephosphorylation (see below).

Surrounding these ions is a Y-shaped cleft with three grooves: a hydrophobic, an acidic, and a C-terminal groove. When PP1 is not bonded to any other subunit, it is not particularly specific. However, when it bonds to the second subunit of myosin phosphatase, MYPT1 (MW ~130 kDa), this catalytic cleft changes configuration. This results in a dramatic increase in myosin specificity. [1] Thus, it is clear that MYPT1 has great regulatory power over PP1 and myosin phosphatase, even without the presence of other activators or inhibitors.

The third subunit, M20 (not to be confused with MLC20, the critical regulatory subunit of myosin), is the smallest and most mysterious subunit. Currently little is known about M20, except that it is not necessary for catalysis, as removing the subunit does not affect turnover or selectivity. [1] While some believe it could have regulatory function, nothing has been determined yet. [2]

Mechanism

The mechanism of removing the phosphate from Ser-19 is very similar to other dephosphorylation reactions in the cell, such as the activation of glycogen synthase. Myosin's regulatory subunit MLC20 binds to both the hydrophobic and acid grooves of PP1 and MYPT1, the regulatory site on myosin phosphatase. [1] [8] Once in the proper configuration, both the phyosphorylated serine and a free water molecule are stabilized by the hydrogen-bonding residues in the active site, as well as the positively charged ions (which interact strongly with the negative phosphate group). His-125 (on myosin phosphatase) donates a proton to Ser-19 MLC20), and the water molecule attacks the phosphorus atom. After shuffling protons to stabilize (which happens rapidly compared to the attack on phosphorus), the phosphate and alcohol are formed, and both leave the active site.

The mechanism of PP1 for myosin phosphatase, with critical enzyme residues shown. The substrates and products are bold and in red, and the manganese ions are in blue. The alcohol group shown on myosin after dephosphorylation is the alcohol on Ser-19. PP1 mechanism for myosin phosphatase.png
The mechanism of PP1 for myosin phosphatase, with critical enzyme residues shown. The substrates and products are bold and in red, and the manganese ions are in blue. The alcohol group shown on myosin after dephosphorylation is the alcohol on Ser-19.

Regulation and Human Health

The regulatory pathways of MLC kinase have been well-established, but until the late 1980s, it was assumed that myosin phosphatase was not regulated, and contraction/relaxation was entirely dependent on MLC kinase activity. [2] However, since the 1980s, the inhibiting effect of rho-associated protein kinase has been discovered and thoroughly investigated. [11] RhoA GTP activates Rho-kinase, which phosphorylates the MYPT1 at two major inhibitory sites, Thr-696 and Thr-866. [12] [13] This fully demonstrates the value of the MYPT1, not only to increase reaction rate and specificity, but also to greatly slow down the reaction. However, when telokin is added, it effectively undoes the effect of Rho-kinase, even though it does not dephosphorylate MYPT1. [12]

One other proposed regulatory strategy involves arachidonic acid. When arachidonic acid is added to tensed muscle tissue, the acid decreases the rate of dephosphorylation (and thus relaxation) of myosin. However, it is unclear how arachidonic acid functions as an inhibitor. [4] Two competing theories are that either arachidonic acid acts as a co-messenger in the rho-kinase cascade mentioned above, or that it binds to the c-terminal of MYPT1. [4]

When the regulatory systems of myosin phosphatase begin to fail, there can be major health consequences. Since smooth muscle is found in the respiratory, circulatory, and reproductive systems of humans (as well as other places), if the smooth muscle can no longer relax because of faulty regulation, then a wide number of problems ranging from asthma, hypertension, and erectile dysfunction can result. [4] [14]

See also

Related Research Articles

A protein phosphatase is a phosphatase enzyme that removes a phosphate group from the phosphorylated amino acid residue of its substrate protein. Protein phosphorylation is one of the most common forms of reversible protein posttranslational modification (PTM), with up to 30% of all proteins being phosphorylated at any given time. Protein kinases (PKs) are the effectors of phosphorylation and catalyse the transfer of a γ-phosphate from ATP to specific amino acids on proteins. Several hundred PKs exist in mammals and are classified into distinct super-families. Proteins are phosphorylated predominantly on Ser, Thr and Tyr residues, which account for 79.3, 16.9 and 3.8% respectively of the phosphoproteome, at least in mammals. In contrast, protein phosphatases (PPs) are the primary effectors of dephosphorylation and can be grouped into three main classes based on sequence, structure and catalytic function. The largest class of PPs is the phosphoprotein phosphatase (PPP) family comprising PP1, PP2A, PP2B, PP4, PP5, PP6 and PP7, and the protein phosphatase Mg2+- or Mn2+-dependent (PPM) family, composed primarily of PP2C. The protein Tyr phosphatase (PTP) super-family forms the second group, and the aspartate-based protein phosphatases the third. The protein pseudophosphatases form part of the larger phosphatase family, and in most cases are thought to be catalytically inert, instead functioning as phosphate-binding proteins, integrators of signalling or subcellular traps. Examples of membrane-spanning protein phosphatases containing both active (phosphatase) and inactive (pseudophosphatase) domains linked in tandem are known, conceptually similar to the kinase and pseudokinase domain polypeptide structure of the JAK pseudokinases. A complete comparative analysis of human phosphatases and pseudophosphatases has been completed by Manning and colleagues, forming a companion piece to the ground-breaking analysis of the human kinome, which encodes the complete set of ~536 human protein kinases.

<span class="mw-page-title-main">Smooth muscle</span> Involuntary non-striated muscle

Smooth muscle is an involuntary non-striated muscle, so-called because it has no sarcomeres and therefore no striations. It is divided into two subgroups, single-unit and multiunit smooth muscle. Within single-unit muscle, the whole bundle or sheet of smooth muscle cells contracts as a syncytium.

<span class="mw-page-title-main">Protein kinase A</span> Family of enzymes

In cell biology, protein kinase A (PKA) is a family of serine-threonine kinase whose activity is dependent on cellular levels of cyclic AMP (cAMP). PKA is also known as cAMP-dependent protein kinase. PKA has several functions in the cell, including regulation of glycogen, sugar, and lipid metabolism. It should not be confused with 5'-AMP-activated protein kinase.

In biochemistry, dephosphorylation is the removal of a phosphate (PO43−) group from an organic compound by hydrolysis. It is a reversible post-translational modification. Dephosphorylation and its counterpart, phosphorylation, activate and deactivate enzymes by detaching or attaching phosphoric esters and anhydrides. A notable occurrence of dephosphorylation is the conversion of ATP to ADP and inorganic phosphate.

<span class="mw-page-title-main">Glycogen synthase</span> Enzyme class, includes all types of glycogen/starch synthases

Glycogen synthase is a key enzyme in glycogenesis, the conversion of glucose into glycogen. It is a glycosyltransferase that catalyses the reaction of UDP-glucose and n to yield UDP and n+1.

<span class="mw-page-title-main">Telokin</span> Protein domain

Telokin is an abundant protein found in smooth-muscle. It is identical to the C-terminus of myosin light-chain kinase. Telokin may play a role in the stabilization of unphosphorylated smooth-muscle myosin filaments. Because of its origin as the C-terminal end of smooth muscle myosin light chain kinase, it is called "telokin".

<span class="mw-page-title-main">Myosin light-chain kinase</span> Class of kinase enzymes

Myosin light-chain kinase also known as MYLK or MLCK is a serine/threonine-specific protein kinase that phosphorylates a specific myosin light chain, namely, the regulatory light chain of myosin II.

<span class="mw-page-title-main">Nicorandil</span> Chemical compound

Nicorandil is a vasodilatory drug used to treat angina.

<span class="mw-page-title-main">ROCK1</span> Protein

ROCK1 is a protein serine/threonine kinase also known as rho-associated, coiled-coil-containing protein kinase 1. Other common names are ROKβ and P160ROCK. ROCK1 is a major downstream effector of the small GTPase RhoA and is a regulator of the actomyosin cytoskeleton which promotes contractile force generation. ROCK1 plays a role in cancer and in particular cell motility, metastasis, and angiogenesis.

<span class="mw-page-title-main">Myosin light chain</span> Small polypeptide subunit of myosin

A myosin light chain is a light chain of myosin. Myosin light chains were discovered by Chinese biochemist Cao Tianqin when he was a graduate student at the University of Cambridge in England.

<span class="mw-page-title-main">PPP1R12A</span> Protein-coding gene in the species Homo sapiens

Protein phosphatase 1 regulatory subunit 12A is an enzyme that in humans is encoded by the PPP1R12A gene.

<span class="mw-page-title-main">MYH10</span> Protein-coding gene in the species Homo sapiens

Myosin-10 also known as myosin heavy chain 10 or non-muscle myosin IIB (NM-IIB) is a protein that in humans is encoded by the MYH10 gene. Non-muscle myosins are expressed in a wide variety of tissues, but NM-IIB is the only non-muscle myosin II isoform expressed in cardiac muscle, where it localizes to adherens junctions within intercalated discs. NM-IIB is essential for normal development of cardiac muscle and for integrity of intercalated discs. Mutations in MYH10 have been identified in patients with left atrial enlargement.

<span class="mw-page-title-main">MYL2</span> Protein-coding gene in the species Homo sapiens

Myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC-2) also known as the regulatory light chain of myosin (RLC) is a protein that in humans is encoded by the MYL2 gene. This cardiac ventricular RLC isoform is distinct from that expressed in skeletal muscle (MYLPF), smooth muscle (MYL12B) and cardiac atrial muscle (MYL7).

<span class="mw-page-title-main">PPP1R14A</span> Protein found in humans

Protein phosphatase 1 regulatory subunit 14A also known as CPI-17 is a protein that in humans is encoded by the PPP1R14A gene.

<span class="mw-page-title-main">PPP1R12B</span> Protein-coding gene in the species Homo sapiens

Protein phosphatase 1 regulatory subunit 12B is an enzyme that in humans is encoded by the PPP1R12B gene.

Fasudil (INN) is a potent Rho-kinase inhibitor and vasodilator. Since it was discovered, it has been used for the treatment of cerebral vasospasm, which is often due to subarachnoid hemorrhage, as well as to improve the cognitive decline seen in stroke patients. It has been found to be effective for the treatment of pulmonary hypertension. It has been demonstrated that fasudil could improve memory in normal mice, identifying the drug as a possible treatment for age-related or neurodegenerative memory loss.

<span class="mw-page-title-main">Rho-associated protein kinase</span>

Rho-associated protein kinase (ROCK) is a kinase belonging to the AGC family of serine-threonine specific protein kinases. It is involved mainly in regulating the shape and movement of cells by acting on the cytoskeleton.

<span class="mw-page-title-main">Protein phosphatase 1</span>

Protein phosphatase 1 (PP1) belongs to a certain class of phosphatases known as protein serine/threonine phosphatases. This type of phosphatase includes metal-dependent protein phosphatases (PPMs) and aspartate-based phosphatases. PP1 has been found to be important in the control of glycogen metabolism, muscle contraction, cell progression, neuronal activities, splicing of RNA, mitosis, cell division, apoptosis, protein synthesis, and regulation of membrane receptors and channels.

Ceramide-activated protein phosphatases (CAPPs) are a group of enzymes that are activated by the lipid second messenger ceramide. Known CAPPs include members of the protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) families. CAPPs are a subset of intracellular serine/threonine phosphatases. Each CAPP consists of a catalytic subunit which confers phosphatase activity and a regulatory subunit which confers substrate specificity. CAPP involvement has been implicated in glycogen metabolism, apoptotic pathways related to cancer and other cellular pathways related to Alzheimer’s disease.

<span class="mw-page-title-main">Calponin 1</span> Protein-coding gene in the species Homo sapiens

Calponin 1 is a basic smooth muscle protein that in humans is encoded by the CNN1 gene.

References

  1. 1 2 3 4 5 Terrak, Mohammed; Kerff, Frederic; et al. (June 17, 2004). "Structural Basis of Protein Phosphatase 1 Regulation". Nature. 429 (6993): 780–4. Bibcode:2004Natur.429..780T. doi: 10.1038/nature02582 . PMID   15164081.
  2. 1 2 3 Hartshorne, DJ; Ito, M (May 1998). "Myosin Light Chain Phosphatase: Subunit Composition, Interactions and Regulation". J Muscle Res Cell Motil. 19 (4): 325–41. doi:10.1023/A:1005385302064. PMID   9635276. S2CID   27448238.
  3. ↑ Page 174 in: The vascular smooth muscle cell: molecular and biological responses to the extracellular matrix. Authors: Stephen M. Schwartz, Robert P. Mecham. Editors: Stephen M. Schwartz, Robert P. Mecham. Contributors: Stephen M. Schwartz, Robert P. Mecham. Publisher: Academic Press, 1995. ISBN   0-12-632310-0, ISBN   978-0-12-632310-8
  4. 1 2 3 4 Webb, R. Clinton (November 2003). "Smooth Muscle Contraction and Relaxation". Advances in Physiology Education. 27 (4): 201–6. doi:10.1152/advan.00025.2003. PMID   14627615.
  5. ↑ Hurley, Thomas; Yang, Jie; et al. (July 18, 2007). "Structural Basis for Regulation of Protein Phosphatase 1 by Inhibitor-2". J. Biol. Chem. 282 (39): 28874–83. doi: 10.1074/jbc.m703472200 . PMID   17636256.
  6. ↑ Cohen, Patricia T. W. (January 15, 2002). "Protein Phosphatase 1-Targeted in Many Directions". J Cell Sci. 115 (2): 780–4. doi:10.1242/jcs.115.2.241. PMID   11839776.
  7. ↑ Fujioka, M; Takahashi, N (April 1, 1998). "A New Isoform of Human Myosin Phosphatase Targeting/Regulatory Subunit (MYPT2): cDNA Cloning, Tissue Expression, and Chromosomal Mapping". Genomics. 49 (1): 325–41. doi:10.1006/geno.1998.5222. PMID   9570949.
  8. ↑ Gomperts, Bastein D. (August 19, 2009). Signal Transduction: 2nd Edition. London: Academic Press. ISBN   978-0123694416.
  9. ↑ Shi, Yigong (October 30, 2009). "Serine/Threonine Phosphatases: Mechanism through Structure". Cell. 139 (3): 468–84. doi: 10.1016/j.cell.2009.10.006 . PMID   19879837. S2CID   13903804.
  10. ↑ Lee, Ernest Y.C.; Zhang, Lifang; et al. (March 15, 1999). "Phosphorylase Phosphatase: New Horizons for an Old Enzyme". Frontiers in Bioscience. 4 (1–3): 270–85. doi: 10.2741/lee . PMID   10077543 . Retrieved March 9, 2015.
  11. ↑ Wang, Yuepeng; Riddick, Nadeen; et al. (February 27, 2009). "ROCK Isoform Regulation of Myosin Phosphatase and Contractility in Vascular Smooth Muscle Cells". Circ. Res. 104 (4): 531–40. doi:10.1161/circresaha.108.188524. PMC   2649695 . PMID   19131646.
  12. 1 2 Khromov, ES; Momotani, K.; et al. (April 27, 2012). "Molecular Mechanism of Telokin-Mediated Disinhibition of Myosin Light Chain Phosphatase and cAMP/cGMP-Induced Relaxation of Gastrointestinal Smooth Muscle". J Biol Chem. 287 (25): 20975–85. doi: 10.1074/jbc.m112.341479 . PMC   3375521 . PMID   22544752.
  13. ↑ Somlyo, Andrew P.; Somlyo, Avril V. (November 10, 1999). "Signal Transduction by G-Proteins, Rho-Kinase and Protein Phosphatase to Smooth Muscle and Non-Muscle Myosin II". Journal of Physiology. 522 (2): 177–85. doi:10.1111/j.1469-7793.2000.t01-2-00177.x. PMC   2269761 . PMID   10639096.
  14. ↑ Aguilar, Hector; Mitchell, B.F. (May 7, 2010). "Physiological Pathways and Molecular Mechanisms Regulating Uterine Contractility". Human Reproduction Update. 16 (6): 725–44. doi: 10.1093/humupd/dmq016 . PMID   20551073.

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