Serine / Threonine Protein Phosphatases Type 1, 2A and 2C in Vertebrate Retinae

The PKC1 gene of Saccharomyces cerevisiae encodes a homolog of mammalian protein kinase C that is required for yeast cell growth. Loss of PKC1 function results in cell lysis due to an inability to remodel the cell wall properly during growth. The PKC1 gene has been proposed to regulate a bifurcated pathway, on one branch of which function four putative protein kinases that catalyze a linear cascade of protein phosphorylation culminating in the activation of the mitogen-activated protein kinase homolog, Mpk1p. Here we describe two genes whose overexpression suppress both an mpk1 delta mutation and a pkc1 delta mutation. One of these genes is identical to the previously identified PPZ2 gene. The PPZ2 gene is predicted to encode a type 1-related protein phosphatase and is functionally redundant with a closely related gene, designated PPZ1. Deletion of both PPZ1 and PPZ2 resulted in a temperature-dependent cell lysis defect similar to that observed for bck1 delta, mkk1,2 delta, or mpk1 delta mutants. However, ppz1,2 delta mpk1 delta triple mutants displayed a cell lysis defect at all temperatures. The additivity of the ppz1,2 delta defect with the mpk1 delta defect, combined with the results of genetic epistasis experiments, suggested either that the PPZ1- and PPZ2-encoded protein phosphatases function on a branch of the PKC1-mediated pathway different from that defined by the protein kinases or that they play an auxiliary role in the pathway. The other suppressor gene, designated BCK2 (for bypass of C kinase), is predicted to encode a 92-kDa protein that is rich in serine and threonine residues. Genetic interactions between BCK2 and other pathway components suggested that BCK2 functions on a common pathway branch with PPZ1 and PPZ2.

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Involvement of protein phosphatases 1 and 2A in the control of M phase-promoting factor activity in starfish.

The Journal of Cell Biology ◽

10.1083/jcb.109.6.3347 ◽

1989 ◽

Vol 109 (6) ◽

pp. 3347-3354 ◽

Cited By ~ 78

Author(s):

A Picard ◽

J P Capony ◽

D L Brautigan ◽

M Dorée

Keyword(s):

Okadaic Acid ◽

Protein Phosphatases ◽

Germinal Vesicle ◽

Histone H1 ◽

Specific Inhibition ◽

Factor Activity ◽

Microtubule Network ◽

Germinal Vesicle Breakdown ◽

M Phase

Specific inhibition of types 1 and 2A protein phosphatases by microinjection of okadaic acid (OA) into starfish oocytes induced germinal vesicle breakdown and activation of M phase-promoting factor (MPF) and histone H1 kinase. The effects were evident in immature oocytes arrested at first meiotic prophase as well as in fully mature oocytes arrested at the pronucleus stage. In addition, MPF and histone H1 kinase were stabilized for several hours and protected from inactivation by inhibition of type 1 protein phosphatases with either OA or specific anti-phosphatase antibodies. Microinjection of okadaic acid was associated with unusual changes of the microtubule network, including the disappearance of spindles and extension of the cytoplasmic array of microtubules. MPF activation after OA injection was associated with dephosphorylation of phosphothreonine and phosphoserine residues in cdc2, showing that neither type 1 nor 2A protein phosphatases catalyzes these dephosphorylations. The effects of OA on MPF activation and inactivation appeared to involve the cyclin subunit. OA did not induce MPF activation in the absence of protein synthesis and it prevented degradation of cyclin. Therefore protein phosphatases types 1 and 2A appear to be involved in activation and inactivation of MPF involving mechanisms that operate after cyclin synthesis and before its degradation.

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Distinct, essential roles of type 1 and 2A protein phosphatases in the control of the fission yeast cell division cycle

Cell ◽

10.1016/0092-8674(90)90173-c ◽

1990 ◽

Vol 63 (2) ◽

pp. 405-415 ◽

Cited By ~ 174

Author(s):

Noriyuki Kinoshita ◽

Hiroyuki Ohkura ◽

Mitsuhiro Yanagida

Keyword(s):

Cell Division ◽

Yeast Cell ◽

Fission Yeast ◽

Protein Phosphatases ◽

Cell Division Cycle ◽

Fission Yeast Cell

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Inhibitory effect of okadaic acid on the p-nitrophenyl phosphate phosphatase activity of protein phosphatases

Biochemical Journal ◽

10.1042/bj2750233 ◽

1991 ◽

Vol 275 (1) ◽

pp. 233-239 ◽

Cited By ~ 119

Author(s):

A Takai ◽

G Mieskes

Keyword(s):

Phosphatase Activity ◽

Okadaic Acid ◽

Protein Phosphatase ◽

Protein Phosphatases ◽

Inhibitory Effect ◽

Enzyme Concentration ◽

Alkaline Phosphatases ◽

Nitrophenyl Phosphate ◽

Type 1 Phosphatase

The phosphatase activities of type 2A, type 1 and type 2C protein phosphatase preparations were measured against p-nitrophenyl phosphate (pNPP), a commonly used substrate for alkaline phosphatases. Of the three types of phosphatase examined, the type 2A phosphatase exhibited an especially high pNPP phosphatase activity (119 +/- 8 mumol/min per mg of protein; n = 4). This activity was strongly inhibited by pico- to nano-molar concentrations of okadaic acid, a potent inhibitor of type 2A and type 1 protein phosphatases that has been shown to have no effect on alkaline phosphatases. The dose-inhibition relationship was markedly shifted to the right and became steeper by increasing the concentration of the enzyme, as predicted by the kinetic theory for tightly binding inhibitors. The enzyme concentration estimated by titration with okadaic acid agreed well with that calculated from the protein content and the molecular mass for type 2A phosphatase. These results strongly support the idea that the pNPP phosphatase activity is intrinsic to type 2A protein phosphatase and is not due to contamination by alkaline phosphatases. pNPP was also dephosphorylated, but at much lower rates, by type 1 phosphatase (6.4 +/- 8 nmol/min per mg of protein; n = 4) and type 2C phosphatase (1.2 +/- 3 nmol/min per mg of protein; n = 4). The pNPP phosphatase activity of the type 1 phosphatase preparation shows a susceptibility to okadaic acid similar to that of its protein phosphatase activity, whereas it was interestingly very resistant to inhibitor 2, an endogenous inhibitory factor of type 1 protein phosphatase. The pNPP phosphatase activity of type 2C phosphatase preparation was not affected by up to 10 microM-okadaic acid.

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Protein phosphatases type 1 and type 2 in Drosophila melanogaster

Comparative Biochemistry and Physiology Part B Comparative Biochemistry ◽

10.1016/0305-0491(87)90402-0 ◽

1987 ◽

Vol 87 (4) ◽

pp. 857-861 ◽

Cited By ~ 1

Author(s):

Viktor Dombrádi ◽

Peter Friedrich ◽

Georg Bot

Keyword(s):

Drosophila Melanogaster ◽

Protein Phosphatases

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Reconstitution of calyculin-inhibited K-Cl cotransport in dog erythrocyte ghosts by exogenous PP-1

AJP Cell Physiology ◽

10.1152/ajpcell.1996.270.3.c898 ◽

1996 ◽

Vol 270 (3) ◽

pp. C898-C902 ◽

Cited By ~ 38

Author(s):

T. Krarup ◽

P. B. Dunham

Keyword(s):

Protein Phosphatase ◽

Direct Evidence ◽

Potent Inhibitor ◽

Protein Phosphatases ◽

Calyculin A ◽

Osmotic Swelling ◽

Maximal Activation ◽

Resealed Ghosts ◽

K Transport

Osmotic swelling of dog and other mammalian erythrocytes activates Cl-dependent K transport, K-Cl cotransport. This activation can be abolished by nanomolar concentrations of calyculin, a potent inhibitor of serine-threonine protein phosphatases. Therefore, K-Cl cotransport is probably activated by dephosphorylation by a type 1 and/or type 2A protein phosphatase (PP-1 and PP-2A, respectively). This was tested directly by incorporating exogenous protein phosphatases into resealed ghosts made from dog erythrocytes previously exposed to calyculin. K-Cl cotransport was nearly completely inhibited in the ghosts. Incorporation of PP-1 reconstituted K-Cl cotransport. Maximal reconstitution was up to 90% of the control flux in the ghosts and 0.1 U PP-1/ml lysate gave half-maximal reconstitution of cotransport. In contrast, PP-2A had no effect. This result with PP-1 provides direct evidence that K-Cl cotransport is activated by PP-1 in dog erythrocytes. Half-maximal activation of K-Cl cotransport required approximately 180 molecules of PP-1 per ghost.

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Inhibitory effect of okadaic acid derivatives on protein phosphatases. A study on structure-affinity relationship

Biochemical Journal ◽

10.1042/bj2840539 ◽

1992 ◽

Vol 284 (2) ◽

pp. 539-544 ◽

Cited By ~ 129

Author(s):

A Takai ◽

M Murata ◽

K Torigoe ◽

M Isobe ◽

G Mieskes ◽

...

Keyword(s):

Okadaic Acid ◽

Structural Changes ◽

Protein Phosphatases ◽

Inhibitory Effect ◽

Chemical Processes ◽

Hydroxyl Group ◽

Confidence Limits ◽

X Ray ◽

Structural Modifications

The effect of structural modifications of okadaic acid (OA), a polyether C38 fatty acid, was studied on its inhibitory activity toward type 1 and type 2A protein phosphatases (PP1 and PP2A) by using OA derivatives obtained either by isolation from natural sources or by chemical processes. The dissociation constant (Ki) for the interaction of OA with PP2A was estimated to be 30 (26-33) nM [median (95% confidence limits)]. The OA derivatives used and their affinity for PP2A, expressed as Ki (in brackets) were as follows: 35-methyl-OA (DTX1) [19 (12-25) pM], OA-9,10-episulphide (acanthifolicin) [47 (25-60) pM], 7-deoxy-OA [69 (31-138) pM], 14,15-dihydro-OA [315 (275-360) pM], 2-deoxy-OA [899 (763-1044) pM], 7-O-palmitoyl-OA [greater than 100 nM], 7-O-palmitoyl-DTX1 [greater than 100 nM], methyl okadate [much greater than 100 nM], 2-oxo-decarboxy-OA [much greater than 100 nM] and the C-15-C-38 fragment of OA [much greater than 100 nM]. The sequence of the affinity of these derivatives for PP1 was essentially the same as that observed with PP2A, although the absolute values of Ki were very different for the enzymes. The inhibitory effect of OA on PP2A was reversed by applying a murine monoclonal antibody against OA, which recognizes modifications of the 7-hydroxyl group of the OA molecule. It has been shown by n.m.r. spectroscopy and X-ray analysis that one end (C-1-C-24) of the OA molecule assumes a circular conformation. The present results suggest the importance of the conformation for the inhibitory action of OA on the protein phosphatases. The ratios of the Ki values for PP1 to that for PP2A, which were within the range 10(3)-10(4), tended to be smaller for the derivatives with lower affinity, indicating that the structural changes in OA impaired the affinity for PP2A more strongly than that for PP1.

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