Purification and characterization of protein phosphatase 2A from petals of the tulip Tulipa gesnerina

The holoenzyme of protein phosphatase (PP) from tulip petals was purified by using hydrophobic interaction, anion exchange and microcystin affinity chromatography to analyze activity towards p-nitrophenyl phosphate (p-NPP). The catalytic subunit of PP was released from its endogenous regulatory subunits by ethanol precipitation and further purified. Both preparations were characterized by immunological and biochemical approaches to be PP2A. On SDS-PAGE, the final purified holoenzyme preparation showed three protein bands estimated at 38, 65, and 75 kDa while the free catalytic subunit preparation showed only the 38 kDa protein. In both preparations, the 38 kDa protein was identified immunologically as the catalytic subunit of PP2A by using a monoclonal antibody against the PP2A catalytic subunit. The final 623- and 748- fold purified holoenzyme and the free catalytic preparations, respectively, exhibited high sensitivity to inhibition by 1 nM okadaic acid when activity was measured with p-NPP. The holoenzyme displayed higher stimulation in the presence of ammonium sulfate than the free catalytic subunit did by protamine, thereby suggesting different enzymatic behaviors.     

Assembly and structure of protein phosphatase 2A

Protein phosphatase 2A (PP2A) represents a conserved family of important protein serine/threonine phosphatases in species ranging from yeast to human. The PP2A core enzyme comprises a scaffold subunit and a catalytic subunit. The heterotrimeric PP2A holoenzyme consists of the core enzyme and a variable regulatory subunit. The catalytic subunit of PP2A is subject to reversible methylation, medi-ated by two conserved enzymes. Both the PP2A core and holoenzymes are regulated through interac-tion with a large n...     

Association of the type 1 protein phosphatase PP1 with the A-kinase anchoring protein AKAP220

The cyclic AMP (cAMP)-dependent protein kinase (PKA) and the type 1 protein phosphatase (PP1) are broad-specificity signaling enzymes with opposing actions that catalyze changes in the phosphorylation state of cellular proteins. Subcellular targeting to the vicinity of preferred substrates is a means of restricting the specificity of each enzyme [1] [2]. Compartmentalization of the PKA holoenzyme is mediated through association of the regulatory subunits with A-kinase anchoring proteins (AKAPs), whereas a diverse family of phosphatase-targeting subunits directs the location of the PP1 catalytic subunit (PP1c) [3] [4]. Here, we demonstrate that the PKA-anchoring protein, AKAP220, binds PP1c with a dissociation constant (KD) of 12.1 +/- 4 nM in vitro. Immunoprecipitation of PP1 from cell extracts resulted in a 10.4 +/- 3.8-fold enrichment of PKA activity. AKAP220 co-purified with PP1c by affinity chromatography on microcystin sepharos Immunocytochemical analysis demonstrated that the kinase, the phosphatase and the anchoring protein had distinct but overlapping staining patterns in rat hippocampal neurons. Collectively, these results provide the first evidence that AKAP220 is a multivalent anchoring protein that maintains a signaling scaffold of PP1 and the PKA holoenzyme.     

Structure of the protein phosphatase 2A holoenzyme

Protein Phosphatase 2A (PP2A) plays an essential role in many aspects of cellular physiology. The PP2A holoenzyme consists of a heterodimeric core enzyme, which comprises a scaffolding subunit and a catalytic subunit, and a variable regulatory subunit. Here we report the crystal structure of the heterotrimeric PP2A holoenzyme involving the regulatory subunit B'/B56/PR61. Surprisingly, the B'/PR61 subunit has a HEAT-like (huntingtin-elongation-A subunit-TOR-like) repeat structure, similar to that of the scaffolding subunit. The regulatory B'/B56/PR61 subunit simultaneously interacts with the catalytic subunit as well as the conserved ridge of the scaffolding subunit. The carboxyterminus of the catalytic subunit recognizes a surface groove at the interface between the B'/B56/PR61 subunit and the scaffolding subunit. Compared to the scaffolding subunit in the PP2A core enzyme, formation of the holoenzyme forces the scaffolding subunit to undergo pronounced conformational rearrangements. This structure reveals significant ramifications for understanding the function and regulation of PP2A.     

A RNA interference screen identifies the protein phosphatase 2A subunit PR55gamma as a stress-sensitive inhibitor of c-SRC

Protein Phosphatase type 2A (PP2A) represents a family of holoenzyme complexes with diverse biological activities. Specific holoenzyme complexes are thought to be deregulated during oncogenic transformation and oncogene-induced signaling. Since most studies on the role of this phosphatase family have relied on the use of generic PP2A inhibitors, the contribution of individual PP2A holoenzyme complexes in PP2A-controlled signaling pathways is largely unclear. To gain insight into this, we have constructed a set of shRNA vectors targeting the individual PP2A regulatory subunits for suppression by RNA interference. Here, we identify PR55gamma and PR55delta as inhibitors of c-Jun NH(2)-terminal kinase (JNK) activation by UV irradiation. We show that PR55gamma binds c-SRC and modulates the phosphorylation of serine 12 of c-SRC, a residue we demonstrate to be required for JNK activation by c-SRC. We also find that the physical interaction between PR55gamma and c-SRC is sensitive to UV irradiation. Our data reveal a novel mechanism of c-SRC regulation whereby in response to stress c-SRC activity is regulated, at least in part, through loss of the interaction with its inhibitor, PR55gamma.     

Sperm PP1gamma2 is regulated by a homologue of the yeast protein phosphatase binding protein sds22

Serine/threonine phosphatase PP1gamma2 is a testis-specific protein phosphatase isoform in spermatozoa. This enzyme appears to play a key role in motility initiation and stimulation. Catalytic activity of PP1gamma2 is higher in immotile compared with motile spermatozoa. Inhibition of PP1gamma2 activity causes both motility initiation and motility stimulation. Protein phosphatases, in general, are regulated by their binding proteins. The objective of this article is to understand the mechanisms by which PP1gamma2 is regulated, first by identifying its regulatory proteins. We had previously shown that a portion of bovine sperm PP1gamma2 is present in the cytosolic fraction of sperm sonicates. We purified PP1gamma2 from soluble bovine sperm extracts by immunoaffinity chromatography. Gel electrophoresis of the purified enzyme showed that it was complexed to a protein 43 M(r) x 10(-3) in size. Microsequencing revealed that this protein is a mammalian homologue of sds22, which is a yeast PP1 binding protein. Phosphatase activity measurements showed that PP1gamma2 complexed to sds22 is catalytically inactive. The complex cannot be activated by limited proteolysis. The complex is unable to bind to microcystin sepharose. This suggests that sds22 may block the microcystin binding site in PP1gamma2. A proportion of PP1gamma2 in sperm extracts, which is presumably not complexed to sds22, is catalytically active. Fluorescence immunocytochemistry was used to determine the intrasperm localization of PP1gamma2 and sds22. Both proteins are present in the tail. They are also present in distinct locations in the head. Our data suggest that PP1gamma2 binding to sds22 inhibits its catalytic activity. Mechanisms regulating sds22 binding to PP1gamma2 are likely to be important in understanding the biochemical basis underlying development and regulation of sperm function.     

Dilated cardiomyopathy in transgenic mice expressing a mutant A subunit of protein phosphatase 2A

The protein phosphatase 2A (PP2A) holoenzyme consists of a catalytic subunit, C, and two regulatory subunits, A and B. The PP2A core enzyme is composed of subunits A and C. Both the holoenzyme and the core enzyme are similarly abundant in heart tissue. Transgenic mice were generated expressing high levels of a dominant negative mutant of the A subunit (A delta 5) in the heart, skeletal muscle, and smooth muscle that competes with the endogenous A subunit for binding the C subunit but does not bind B subunits. We found that the ratio of core enzyme to holoenzyme was increased in A delta 5-expressing hearts. Importantly, already at day 1 after birth, A delta 5-transgenic mice had an increased heart weight-to-body weight ratio that persisted throughout life. Echocardiographic analysis of A delta 5-transgenic hearts revealed increased end-diastolic and end-systolic dimensions and decreased fractional shortening. In addition, the thickness of the septum and of the left ventricular posterior wall was significantly reduced. On the basis of these findings, we consider the heart phenotype of A delta 5-transgenic mice to be a form of dilated cardiomyopathy that frequently leads to premature death.     

Properties of the gamma subunit of phosphorylase kinase

Enzymatic properties of the isolated, active gamma subunit of phosphorylase kinase were characterized. Kinetic parameters indicated that the gamma subunit binds the substrates MgATP and phosphorylase b as well as the holoenzyme with a Km (MgATP) of 98 microM and a Km (phosphorylase b) of 80 microM at pH 8.2, but maximal velocities are significantly lower than the holoenzyme's. Unlike the gamma-calmodulin complex, the gamma subunit activity is dependent on pH in the range of pH 6.2-9.0, with a ratio of activity at pH 6.8 to activity at pH 8.2 of 0.5-0.6. Calmodulin activates the gamma subunit more at low pH than at high pH. ADP inhibits the gamma subunit in a competitive manner with a Ki of 60 microM. Free Mg2+ stimulates gamma subunit activity 3.5-fold at both pH 6.8 and 8.2. MnATP is equivalent to MgATP as a substrate for the enzyme, but free Mn2+ inhibits gamma subunit activity. Several protein substrates of holophosphorylase kinase were found also to be phosphorylated by the gamma subunit. These included kappa-casein, myelin basic protein, the troponin complex, and troponin T alone. In the troponin complex, the proportion of 32P incorporated by the gamma subunit into troponin I compared with troponin T was not Ca2+ dependent, but with the holoenzyme, this proportion was changed greatly by Ca2+ concentration.     

Generation of active protein phosphatase 2A is coupled to holoenzyme assembly

Protein phosphatase 2A (PP2A) is a prime example of the multisubunit architecture of protein serine/threonine phosphatases. Until substrate-specific PP2A holoenzymes assemble, a constitutively active, but nonspecific, catalytic C subunit would constitute a risk to the cell. While it has been assumed that the severe proliferation impairment of yeast lacking the structural PP2A subunit, TPD3, is due to the unrestricted activity of the C subunit, we recently obtained evidence for the existence of the C subunit in a low-activity conformation that requires the RRD/PTPA proteins for the switch into the active conformation. To study whether and how maturation of the C subunit is coupled with holoenzyme assembly, we analyzed PP2A biogenesis in yeast. Here we show that the generation of the catalytically active C subunit depends on the physical and functional interaction between RRD2 and the structural subunit, TPD3. The phenotype of the tpd3Δ strain is therefore caused by impaired, rather than increased, PP2A activity. TPD3/RRD2-dependent C subunit maturation is under the surveillance of the PP2A methylesterase, PPE1, which upon malfunction of PP2A biogenesis, prevents premature generation of the active C subunit and holoenzyme assembly by counteracting the untimely methylation of the C subunit. We propose a novel model of PP2A biogenesis in which a tightly controlled activation cascade protects cells from untargeted activity of the free catalytic PP2A subunit.     

Purification and properties of Arabidopsis thaliana type 1 protein phosphatase (PP1).

The Arabidopsis thaliana type 1 protein phosphatase (PP1) catalytic subunit was released from its endogenous regulatory subunits by ethanol precipitation and purified by anion exchange and microcystin affinity chromatography. The enzyme was identified by MALDI-TOF mass spectrometry from a tryptic digest of the purified protein as a mixture of PP1 isoforms (TOPP 1-6) indicating that at least 4-6 of the eight known PP1 proteins are expressed in sufficient quantities for purification from A. thaliana suspension cells. The enzyme had a final specific activity of 8950 mU/mg using glycogen phosphorylase a as substrate, had a subunit molecular mass of 35 kDa as determined by SDS-PAGE and behaved as a monomeric protein of approx. 39 kDa on Superose 12 gel filtration chromatography. Similar to the mammalian type 1 protein phosphatases, the A. thaliana enzyme was potently inhibited by Inhibitor-2 (IC(50)=0.65 nM), tautomycin (IC(50)=0.06 nM), microcystin-LR (IC(50)=0.01 nM), nodularin (IC(50)=0.035 nM), calyculin A (IC(50)=0.09 nM), okadaic acid (IC(50)=20 nM) and cantharidin (IC(50)=60 nM). The enzyme was also inhibited by fostriecin (IC(50)=22 microM), NaF (IC(50)=2.1 mM), Pi (IC(50)=9.5 mM), and PPi (IC(50)=0.07 mM). Purification of the free catalytic subunit allowed it to be used to probe protein phosphatase holoenzyme complexes that were enriched on Q-Sepharose and a microcystin-Sepharose affinity matrix and confirmed several proteins to be PP1 targeting subunits.     

Inhibition of protein phosphatase 2A activity by selective electrophile alkylation damage

Protein serine/threonine phosphatase 2A (PP2A) is a critical regulator of numerous cellular signaling processes and a potential target for reactive electrophiles that dysregulate phosphorylation-dependent signal transduction cascades. The predominant cellular form of PP2A is a heterotrimeric holoenzyme consisting of a structural A, a variable B, and a catalytic C subunit. We studied the modification of two purified PP2A holoenzyme complexes (ABalpha(FLAG)C and ABdelta(FLAG)C) with two different thiol-reactive electrophiles, biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine (PEO-IAB) and the biotinamido-4-[4'-(maleimidomethyl)cyclohexanecarboxamido]butane (BMCC). In vivo treatment of HEK 293 cells with these electrophiles resulted in alkylation of all three PP2A subunits. Electrophile treatment of the immunopurified FLAG-tagged holoenzymes produced a concentration-dependent adduction of PP2A subunits, as observed by Western blot analysis. Although both electrophiles labeled all three PP2A subunits, only BMCC inhibited the catalytic activity of both holoenzymes. Alkylation patterns in the A and B subunits were identical for the two electrophiles, but BMCC alkylated four Cys residues in the C subunit that were not labeled by PEO-IAB. Homology between the catalytic subunits of PP1 and PP2A enabled generation of a comparative model structure for the C subunit of PP2A. The model structure provided additional insight into contributions of specific BMCC-Cys adducts to PP2A enzyme inhibition. The results indicate that site selectivity of protein adduction should be a critical determinant of the ability of electrophiles to affect cellular signaling processes.     

Purification and characterization of the glycogen-bound protein phosphatase from rat liver

Glycogen-bound protein phosphatase G from rat liver was transferred from glycogen to beta-cyclodextrin (cycloheptaamylose) linked to Sepharose 6B. After removal of the catalytic subunit and of contaminating proteins with 2 M NaCl, elution with beta-cyclodextrin yielded a single protein on native polyacrylamide gel electrophoresis and two polypeptides (161 and 54 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Several lines of evidence indicate that the latter polypeptides are subunits of the protein phosphatase G holoenzyme. First, these polypeptides were also present, together with the catalytic subunit, in the extensively purified holoenzyme. Also, polyclonal antibodies against these polypeptides were able to bind the holoenzyme. Further, while bound to cyclodextrin-Sepharose, the polypeptides were able to recombine with separately purified type-1 (AMD) catalytic subunit, but not with type-2A (PCS) catalytic subunit. The characteristics of the reconstituted enzyme resembled those of the nonpurified protein phosphatase G. At low dilutions, the spontaneous phosphorylase phosphatase activity of the reconstituted enzyme was about 10 times lower than that of the catalytic subunit, but it was about 1000-fold more resistant to inhibition by the modulator protein (inhibitor-2). In contrast with the free catalytic subunit, the reconstituted enzyme co-sedimented with glycogen, and it was able to activate purified liver glycogen synthase b. Also, the synthase phosphatase activity was synergistically increased by a cytosolic phosphatase and inhibited by physiological concentrations of phosphorylase alpha and of Ca2+.     

Clathrin light chain b is capable of affecting potently a major protein phosphatase from microtubules (MT-PP1)

Clathrin light chain (CL) b purified from bovine brain postmicrotubule supernatant and identified by mass spectrometry potently inhibited a catalytic activity of a major protein phosphatase (PP) that was copurified with microtubules and recognized by antiPP1 antibodies. CLb similarly affected the catalytic subunit and holoenzyme of the PP, little inhibiting the activity of PP2A. Although the CLb from clathrin-coated vesicles was several hundredfold weaker than our purified CLb, the CLb in the postmicrotubule supernatant, independent of whether it was sedimentable or soluble, was as active as the purified CLb. Thus CLb may be a potent regulator of the PP.     

siRNA-mediated depletion of endogenous protein phosphatase 2Acalpha markedly attenuates ceramide-activated protein phosphatase activity in insulin-secreting INS-832/13 cells

The sphingolipid ceramide (CER) and its metabolites have been recognized as important mediators of signal transduction processes leading to a variety of cellular responses, including survival and demise via apoptosis. Accumulating evidence implicates key regulatory roles for intracellularly generated CER in metabolic dysfunction of the islet beta cell. We have previously reported localization of an okadaic (OKA)-sensitive CER-activated protein phosphatase (CAPP) in the islet beta cell. We have also reported immunological identification of the structural A subunit, the regulatory B56alpha subunit, and the catalytic C subunit for CAPP holoenzyme complex in insulin-secreting INS-1 cells. Herein, we provide the first evidence to suggest that siRNA-mediated knockdown of the alpha isoform of the catalytic subunit of PP2Ac (PP2Acalpha) markedly reduces the CAPP activity in INS 832/13 cells. Potential significance of the functional activation of CAPP holoenzyme in the context of lipid-and glucose-induced metabolic dysfunction of the islet beta cell is discussed.     

Overexpression of AtPTPA in Arabidopsis increases protein phosphatase 2A activity by promoting holoenzyme formation and ABA negatively affects holoenzyme formation

AtPTPA is a critical regulator for the holoenzyme assembling of protein phosphatase 2A (PP2A) in Arabidopsis. Characterization of AtPTPA improves our understanding of the function and regulation of PP2A in eukaryotes. Further analysis of AtPTPA-overexpressing plants indicates that AtPTPA increases PP2A activity by promoting PP2A''s AC dimer formation, thereby holoenzyme assembling. Plant hormone abscisic acid (ABA) reduces PP2A enzyme activity by negatively affects PP2A''s AC dimer formation. Therefore, AtPTPA is a positive factor that promotes PP2A holoenzyme assembly, and ABA is a negative factor that prevents PP2A holoenzyme assembly.     

Separation of PP2A core enzyme and holoenzyme with monoclonal antibodies against the regulatory A subunit: abundant expression of both forms in cells.

Protein phosphatase 2A (PP2A) holoenzyme is composed of a catalytic subunit, C, and two regulatory subunits, A and B. The A subunit is rod shaped and consists of 15 nonidentical repeats. According to our previous model, the B subunit binds to repeats 1 through 10 and the C subunit binds to repeats 11 through 15 of the A subunit. Another form of PP2A, core enzyme, is composed only of subunits A and C. It is generally believed that core enzyme does not exist in cells but is an artifact of enzyme purification. To study the structure and relative abundance of different forms of PP2A, we generated monoclonal antibodies against the native A subunit. Two antibodies, 5H4 and 1A12, recognized epitopes in repeat 1 near the N terminus and immunoprecipitated free A subunit and core enzyme but not holoenzyme. Another antibody, 6G3, recognized an epitope in repeat 15 at the C terminus and precipitated only the free A subunit. Monoclonal antibodies against a peptide corresponding to the N-terminal 11 amino acids of the A alpha subunit (designated 6F9) precipitated free A subunit, core enzyme, and holoenzyme. 6F9, but not 5H4, recognized holoenzymes containing either B, B', or B" subunits. These results demonstrate that B subunits from three unrelated gene families all bind to repeat 1 of the A subunit, and the results confirm and extend our model of the holoenzyme. By sequential immunoprecipitations with 5H4 or 1A12 followed by 6F9, core enzyme and holoenzyme in cytoplasmic extracts from 10T1/2 cells were completely separated and they exhibited the expected specificities towards phosphorylase a and retinoblastoma peptide as substrates. Quantitative analysis showed that under conditions which minimized proteolysis and dissociation of holoenzyme, core enzyme represented at least one-third of the total PP2A. We conclude that core enzyme is an abundant form in cells rather than an artifact of isolation. The biological implications of this finding are discussed.     

Increased phosphorylation of a distinct subcellular pool of protein phosphatase, PP1gamma2, during epididymal sperm maturation

The enzyme PP1gamma2 is a testis- and sperm-specific isoform of type 1 protein phosphatase (PP1), and it is the only isoform of PP1 in spermatozoa. The enzyme PP1gamma2 is essential for spermatogenesis and is also a key enzyme in the development and regulation of sperm motility. The carboxy terminus of the enzyme contains a consensus amino acid sequence for phosphorylation by cyclin-dependent kinases. Using antibodies specific to this phosphorylated amino acid sequence domain, we found that phosphorylated PP1gamma2 is present in bovine epididymal spermatozoa. The level of phosphorylated PP1gamma2 is significantly higher in motile caudal compared to immotile caput epididymal spermatozoa. A number of treatments, such as 2-chloro adenosine, cAMP analogues, cAMP phosphodiesterase inhibitors, and calcium, which stimulate sperm motility, did not alter the level of phosphorylated PP1gamma2. However, calyculin A, which is an inhibitor of protein phosphatase subtypes PP1 and PP2A, significantly increases the level of phosphorylated PP1gamma2 in both caput and caudal epididymal spermatozoa. Partial purification by column chromatography showed that phosphorylated PP1gamma2 is catalytically active. Phosphorylated PP1gamma2 is the only spontaneously catalytically active form of the enzyme in caudal sperm extracts. Western blot analysis shows that the enzyme cyclin-dependent kinase 2, one of the enzymes that phosphorylates the consensus domain at the carboxy terminus in PP1 isoforms, is present in spermatozoa. Western blot analysis of proteins extracted from purified head and tail fragments of spermatozoa showed that phosphorylated PP1gamma2 is present predominantly in the sperm head. Fluorescence immunocytochemistry also showed that phosphorylated PP1gamma2 is present predominantly in the posterior region of the sperm head. The distinct subcellular localization and changes in its level during sperm maturation suggest a possible role for sperm phosphorylated PP1gamma2 in signaling events during fertilization.     

Cdc55p-mediated E4orf4 growth inhibition in Saccharomyces cerevisiae is mediated only in part via the catalytic subunit of protein phosphatase 2A

The adenovirus early region 4 open reading frame 4 (E4orf4) protein specifically induces p53-independent cell death of transformed but not normal human cells, suggesting that elucidation of its mechanism may provide important new avenues for cancer therapy. Wild-type E4orf4 and mutants that retain cancer cell toxicity also induce growth inhibition in Saccharomyces cerevisiae, which provides a genetically tractable system for studying E4orf4 function. Interaction with the protein phosphatase 2A (PP2A) B regulatory subunit is required for E4orf4's effects, suggesting that E4orf4 may function by regulating B subunit-containing heterotrimeric PP2A holoenzymes (PP2A(BAC)), which consist of a B subunit complexed with the PP2A structural (A) and catalytic (C) subunits. However, it is not known whether E4orf4-induced growth inhibition requires interaction with the PP2A C subunit or whether E4orf4 might have PP2A B subunit-dependent effects that are independent of PP2A(BAC) holoenzyme formation. To test these possibilities in S. cerevisiae, we disrupted the stable formation of PP2A(BAC) heterotrimers and thus E4orf4/C subunit association by PP2A C subunit point mutations or by deletion of the gene for the PP2A methyltransferase, Ppm1p, and assayed for effects on E4orf4-induced growth inhibition. Our results support a model in which E4orf4 mediates growth inhibition and cell killing both through PP2A(BAC) heterotrimers and through a B regulatory subunit-dependent pathway(s) that is independent of stable complex formation with the PP2A C subunit. They also indicate that Ppm1p has a function other than regulating the assembly of PP2A heterotrimers and suggest that selective PP2A trimer inhibitors and PP6 inhibitors may be useful as adjuvant anticancer therapies.     

Subunit composition and developmental regulation of hepatic protein phosphatase 2A (PP2A)

The prototypical form of the Ser/Thr phosphatase PP2A is a heterotrimeric complex consisting of catalytic subunit (C), and A and B regulatory subunits. C-terminal methylation of PP2A-C influences holoenzyme assembly. Using late gestation development in the rat as an in vivo model of liver growth, we found that PP2A-C protein and activity levels were higher in fetal compared to adult liver extracts. However, unmethylated PP2A-C was much higher in the adult extracts. In MonoQ fractionation, unmethylated C eluted separately from methylated C, which was present predominantly in ABC heterotrimers. Gel filtration chromatography revealed that some unmethylated C was present as free catalytic subunit in adult liver. In addition, a significant proportion of PP2A was in inactive forms that may involve novel regulatory subunits. Our results indicate that methylation of PP2A-C appears to be a primary determinant for the biogenesis of PP2A heterotrimers.     

Myosin phosphatase: Unexpected functions of a long-known enzyme

Myosin phosphatase (MP) holoenzyme is a Ser/Thr specific enzyme, which is the member of protein phosphatase type 1 (PP1) family and composed of a PP1 catalytic subunit (PP1c/PPP1CB) and a myosin phosphatase targeting subunit (MYPT1/PPP1R12A). PP1c is required for the catalytic activity of the holoenzyme, while MYPT1 regulates MP through targeting the holoenzyme to its substrates. Above the well-characterized function of MP, as the major regulator of smooth muscle contractility mediating the dephosphorylation of 20 kDa myosin light chain, accumulating data support its role in other, non-contractile functions. In this review, we summarize the scaffold function of MP holoenzyme and its roles in processes such as cell cycle, development, gene expression regulation and neurotransmitter release. In particular, we highlight novel interacting proteins of MYPT1 and pathophysiological functions of MP relevant to tumorigenesis, insulin resistance and neurodegenerative disorders.This article is part of a Special Issue entitled: Protein Phosphatases as Critical Regulators for Cellular Homeostasis edited by Prof. Peter Ruvolo and Dr. Veerle Janssens.