The Protein Phosphatases Invloved in Cellular Regulation. 4. Classification of Two Homogeneous Myosin Light Chain Phosphatases from Smoth Muscle as Protein Phosphatase-2A1 and 2C, and a Homogeneous Protein Phosphatase from Reticulocytes Active on Protein Synthesis Initiation Factor eIF-2 as Protein Phosphatase-2A2

Pato, Mary D.; Adelstein, Robert S.; Crouch, D; Safer, Brian; Ingebritsen, Thomas S.; Cohen, Philip

doi:10.1111/j.1432-1033.1983.tb07360.x

Cited by 82 publications

(7 citation statements)

References 34 publications

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“…PP2A and PP1c both dephosphorylate isolated RLC, but not RLC bound to myosin heavy chain as in smooth muscle heavy meromyosin (Pato et al . ). It is noteworthy that there was significant MLCP activity with smooth muscle heavy meromyosin without MYPT1 in a soluble cytosolic fraction containing PP1cδ from wild‐type tissues, in addition to the significant phosphatase activity in MYPT1‐deficient tissues.…”

Section: Discussionmentioning

confidence: 97%

Constitutive phosphorylation of myosin phosphatase targeting subunit‐1 in smooth muscle

Tsai

Chang

et al. 2014

The Journal of Physiology

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Key pointsr Smooth muscle myosin regulatory light chain (RLC) phosphorylation depends on the relative activities of myosin light chain kinase (MLCK), activated by Ca 2+ -calmodulin, and myosin light chain phosphatase (MLCP).r MYPT1 is a scaffolding protein subunit of MLCP that binds the catalytic subunit PP1cδ and myosin, affects the conformation of PP1cδ for effective inhibition by phosphorylated CPI-17, and is phosphorylated at two sites that inhibit PP1cδ activity.r A conditional knockout of MYPT1 in smooth muscles of adult mice resulted in modest changes in bladder smooth muscle contractile and relaxation responses to KCl or carbachol even though the amount of PP1cδ protein was reduced.r A new a procedure to quantify phosphorylation of MYPT1 showed substantial phosphorylation in wild-type tissues under resting conditions, predicting attenuation of MLCP activity. Reduced PP1cδ activity in MYPT1-deficient tissues may be similar to the attenuated MLCP activity in wild-type tissues resulting from constitutively phosphorylated MYPT1.r In contrast to results obtained with the conditional knockout of MLCK in smooth muscle, MYPT1 is not necessary for smooth muscle function because its loss may not change the effective MLCP activity.Abstract Smooth muscle contraction initiated by myosin regulatory light chain (RLC) phosphorylation is dependent on the relative activities of Ca 2+ -calmodulin-dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). We have investigated the physiological role of the MLCP regulatory subunit MYPT1 in bladder smooth muscle containing a smooth muscle-specific deletion of MYPT1 in adult mice. Deep-sequencing analyses of mRNA and immunoblotting revealed that MYPT1 depletion reduced the amount of PP1cδ with no compensatory changes in expression of other MYPT1 family members. Phosphatase activity towards phosphorylated smooth muscle heavy meromyosin was proportional to the amount of PP1cδ in total homogenates from wild-type or MYPT1-deficient tissues. Isolated MYPT1-deficient tissues from MYPT1 SM−/− mice contracted with moderate differences in response to KCl and carbachol treatments, and relaxed rapidly with comparable rates after carbachol removal and only 1.5-fold slower after KCl removal. Measurements of phosphorylated proteins in the RLC signalling and actin polymerization modules during contractions revealed moderate changes. Using a novel procedure to quantify total phosphorylation of MYPT1 at Thr696 and Thr853, we found substantial phosphorylation in wild-type tissues under resting conditions, predicting attenuation of MLCP activity. Reduced PP1cδ activity in MYPT1-deficient tissues may be similar to the attenuated MLCP activity in wild-type tissues resulting from constitutively phosphorylated MYPT1. Constitutive phosphorylation of MYPT1 Thr696 and Thr853 may thus represent a physiological mechanism acting in concert with agonist-induced MYPT1 phosphorylation to

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Section: Discussionmentioning

confidence: 97%

Constitutive phosphorylation of myosin phosphatase targeting subunit‐1 in smooth muscle

Tsai

Chang

et al. 2014

The Journal of Physiology

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“…It has been shown that both glycogen synthase and eIF-2 can be phosphorylated by protein kinases from rabbit reticulocytes and rabbit muscle that are similar, if not identical, enzymes (DePaoli-Roach et al, 1981). Similarly, although the physiological specificities of particular phosphatases remain to be fully evaluated, it is evident that only a few enzymes can account for the known dephosphorylation activities involved in the regulation of glycogen metabolism, protein synthesis, and other cellular processes (Ingebritsen and Pato et al, 1983;Foulkes et al, 1983). Further studies are directed toward the assessment of eIF-2 phosphorylation state in vivo following ischemia to determine whether such a mechanism could account for the postischemic reduction in brain protein synthesis.…”

Section: Discussionmentioning

confidence: 99%

Changes in Brain Energy Metabolism and Protein Synthesis Following Transient Bilateral Ischemia in the Gerbil

Nowak

Fried

Lust

et al. 1985

Journal of Neurochemistry

178

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The time course of the reduction in brain protein synthesis following transient bilateral ischemia in the gerbil was characterized and compared with changes in a number of metabolites related to brain energy metabolism. The recovery of brain protein synthesis was similar following ischemic periods of 5, 10, or 20 min; in vitro incorporation activity of brain supernatants was reduced to approximately 10% of control at 10 or 30 min recirculation, showed slight recovery at 60 min, and returned to 60% of control activity by 4 h. Protein synthesis activity was indistinguishable from control at 24 h. One minute of ischemia produced no detectable effect on protein synthesis measured after 30 min reperfusion; longer periods of ischemia resulted in progressive inhibition, with 5 min producing the maximal effect. Pentobarbital (50 mg/kg) increased by 1-2 min the threshold ischemic duration required to produce a given effect. Whereas most metabolites recovered quickly following 5 min ischemia, glycogen showed a delayed recovery comparable to that seen for protein synthesis. These results are discussed in relation to possible mechanisms for the coordinate regulation of brain energy metabolism and protein synthesis. An improved method for the fluorimetric measurement of guanine nucleotides is described.

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“…For the purified aortic phosphatase, a Hill coefficient of 0-98-1-02 has been obtained (unpublished observation). In the turkey gizzard, two types of protein phosphatase have been isolated (Fato, Adelstein, Crouch, Safer, Ingebritsen & Cohen, 1983). Therefore, it is possible that the Hill coefficient of less than 1P0 may reflect the heterogeneity of the phosphatase activity in the taenia coli.…”

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confidence: 99%

Effects of okadaic acid on isometric tension and myosin phosphorylation of chemically skinned guinea‐pig taenia coli.

Bialojan

Rüegg

Takai

1988

The Journal of Physiology

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SUMMARY1. In guinea-pig taenia coli skinned with Triton X-100, the marine sponge toxin okadaic acid (OA; 0-1-10 ,UM) produced a dose-dependent enhancement of isometric tension in the presence of low concentrations (01-1 /LM) of Ca".2. The Ca2+-tension relation of the skinned taenia showed a high co-operativity (Hill coefficient, h = 5) in the presence of 02 /LM-calmodulin. The concentration of Ca2+ required to obtain half-maximal tension (ED50) was 1P8 /SM. OA (5/tM) reduced the co-operativity (h = 2 3) and increased the Ca21 sensitivity (ED50 = 0-92 ,gM-Ca21).OA further increased the tension produced with 30 /LM-Ca2 , while it failed to produce any mechanical effect in Ca2+-free solution. When the calmodulin concentration was increased the Ca2+ sensitivity increased as well, but the cooperativity was not affected both in the absence and in the presence of OA.3. The level of myosin phosphorylation was analysed by two-dimensional gel electrophoresis. OA produced an increase in phosphorylated light chains and a concomitant decrease in unphosphorylated light chains. The effect was completely reversed when OA was washed out.4. In solutions containing more than 1 gm1-Ca2+, a third protein band appeared on the gels next to the bands of light chains. OA markedly increased the third band which disappeared when OA and Ca2+ were simultaneously removed.5. OA reversibly slowed down both relaxation and dephosphorylation induced by Ca2+ removal following activation with 30 ,tM-Ca2 . Complete relaxation did not occur in the presence of more than 1 ,uM-OA. The concentration of OA required to produce a 50% reduction (ID60) of the relaxation rate was 78 nM. 6. The phosphatase activity in the taenia extract was inhibited by OA (1-10 /IM) in a dose-dependent manner. The inhibition was well described as a mixed noncompetitive inhibition, and the dose-inhibition relation was shifted to the right when the concentration of substrate (phosphorylated light chains) was increased. The lower and upper limits of the change of ID50 produced by changing the substrate concentration were estimated to be 10 and 165 nM-OA, respectively.7. These results strongly suggest that the tension enhancement and the slow-down of relaxation are both causally related to inhibition of myosin phosphatase activity by OA.

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Cited by 82 publications

References 34 publications

Constitutive phosphorylation of myosin phosphatase targeting subunit‐1 in smooth muscle

Constitutive phosphorylation of myosin phosphatase targeting subunit‐1 in smooth muscle

Changes in Brain Energy Metabolism and Protein Synthesis Following Transient Bilateral Ischemia in the Gerbil

Effects of okadaic acid on isometric tension and myosin phosphorylation of chemically skinned guinea‐pig taenia coli.

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