Effects Of Protein Phosphatases Research Articles

Isoform-specific effects of protein phosphatase 1 on outside-in signalling of integrin αIIbβ3: A story told by genetic manipulation of platelet surrogate cells

Isoform-specific effects of protein phosphatase 1 on outside-in signalling of integrin αIIbβ3: A story told by genetic manipulation of platelet surrogate cells -

Involvement of histone H3 (Ser10) phosphorylation in chromosome condensation without Cdc2 kinase and mitogen-activated protein kinase activation in pig oocytes.

When oocytes resume meiosis, chromosomes start to condense and Cdc2 kinase becomes activated. However, recent findings show that the chromosome condensation does not always correlate with the Cdc2 kinase activity in pig oocytes. The objectives of this study were to examine 1) the correlation between chromosome condensation and histone H3 phosphorylation at serine 10 (Ser10) during the meiotic maturation of pig oocytes and 2) the effects of protein phosphatase 1/2A (PP1/ PP2A) inhibitors on the chromosome condensation and the involvement of Cdc2 kinase, MAP kinase, and histone H3 kinase in this process. The phosphorylation of histone H3 (Ser10) was first detected in the clump of condensed chromosomes at the diakinesis stage and was maintained until metaphase II. The kinase assay showed that histone H3 kinase activity was low in oocytes at the germinal vesicle stage (GV) and increased at the diakinesis stage and that high activity was maintained until metaphase II. Treatment of GV-oocytes with okadaic acid (OA) or calyculin-A (CL-A), the PP1/PP2A inhibitors, induced rapid chromosome condensation with histone H3 (Ser10) phosphorylation after 2 h. Both histone H3 kinase and MAP kinase were activated in the treated oocytes, although Cdc2 kinase was not activated. In the oocytes treated with CL-A and the MEK inhibitor U0126, neither Cdc2 kinase nor MAP kinase were activated and no oocytes underwent germinal vesicle breakdown (GVBD), although histone H3 kinase was still activated and the chromosomes condensed with histone H3 (Ser10) phosphorylation. These results suggest that the phosphorylation of histone H3 (Ser10) occurs in condensed chromosomes during maturation in pig oocytes. Furthermore, the chromosome condensation is correlated with histone H3 kinase activity but not with Cdc2 kinase and MAP kinase activities.

309CHROMOSOME CONDENSATION IS CORRELATED WITH HISTONE H3 PHOSPHORYLATION WITHOUT CDC2 KINASE AND MAP KINASE ACTIVITIES IN PIG OOCYTES

Chromosome condensation is the first step of oocyte maturation. When the oocytes resume meiosis, chromosomes start to condense and Cdc2 kinase becomes activated. However, recent findings show that the chromosome condensation does not always correlate with Cdc2 kinase activity in pig oocytes. The objectives of this study were to examine (1) the correlation between chromosome condensation and histone H3 phosphorylation at serine 10 (Ser10) during meiotic maturation of pig oocytes, and (2) the effects of protein phosphatase 1/2A (PP1/PP2A) inhibitors on the chromosome condensation and the involvement of Cdc2 kinase, MAP kinase and histone H3 kinase in this process. Oocyte-cumulus-granulosa cell complexes (OCGCs) were collected from follicles of 4–6mm in diameter. OCGCs were cultured in modified TCM 199 for different periods of time to obtain oocytes at the germinal vesicle (GV, 0h), diakinesis (18h), metaphase I (24–27h), anaphase I to telophase I (30–33h), and metaphase II (42h) stages. To examine the effects of PP1/PP2A inhibitors on the chromosome condensation, oocyte-cumulus-complexes (OCCs) were cultured in modified TCM 199 with either 2.5μM okadaic acid (OA) or 50nM calyculin A (CL-A) for 0.5, 1, 2, 3, 4 and 6h. To inhibit the MAP kinase activity in the oocytes treated with the PP1/PP2A inhibitor, OCCs were cultured in medium containing CL-A and the MEK inhibitor, U0126 (0.1mM). Morphology of the chromosome and nuclear membrane, and phosphorylation of histone H3 were examined by the immunofluorescent microscopy. In each group 30 oocytes were examined for OA or CL-A and 60 oocytes for CL-A+U0126 treatments. Activities of Cdc2 kinase, MAP kinase and histone H3 kinase were also examined. Phosphorylation of histone H3 (Ser10) was not detected in the oocytes at the GV stage. The phosphorylation was first detected in the clump of condensed chromosomes at the diakinesis stage of prophase I and maintained until metaphase II. The kinase assay also showed that histone H3 kinase activity was low in GV oocytes, increased at the diakinesis stage, and then maintained high activity until metaphase II. PP1/PP2A inhibitors induced rapid chromosome condensation in pig oocytes. Histone H3 phosphorylation (Ser10) became detectable together with the chromosome condensation in the treated oocytes after 2h. After 6h, oocytes had highly condensed chromosomes with phosphorylated histone H3 (81% in CL-A- and 71% in OA-treated oocytes). Both histone H3 kinase and MAP kinase were activated in the treated oocytes, although Cdc2 kinase was not activated. In the oocytes treated with CL-A and U0126, neither Cdc2 kinase nor MAP kinase were activated, although histone H3 kinase was still activated and chromosomes condensed. These results suggest that phosphorylation of histone H3 (Ser10) occurs in condensed chromosomes during maturation in pig oocytes. Futhermore, the chromosome condensation is correlated with histone H3 kinase activity, but not with Cdc2 kinase and MAP kinase activities.

Relationship between biochemical and functional effects of protein phosphatase 1 inhibitors in rabbit cardiac skinned fibers.

Tautomycin (TT) and calyculin A (CyA) are inhibitors of protein phosphatases type 1 and 2 (PP1, PP2). Inhibitors 1 and 2 are specific for PP1, which is the major phosphatase functionally relevant in heart and able to dephosphorylate phospholamban (PLB). TT and CyA maintain PLB in its phosphorylated state, thereby increasing calcium uptake. Rabbit saponin skinned fibers (SF) are used to assess calcium load of the sarcoplasmic reticulum (SR). The present investigation aimed to examine the effects of PP1 inhibitors on SR calcium load assessed by caffeine-induced tension transient (CITT), and to correlate this activity with the PLB phosphorylation state. TT and CyA (100 nm) applied during the uptake phase increased the amplitude of CITT by 10 and 20%, respectively,P<0.05 without effect on the release phase. Both CyA and TT were devoid of calcium sensitizing effect when studied on Triton X-100 SF. After skinning procedure, SF were grinded for biochemical studies. SDS-PAGE electrophoresis and immunoblots using a monoclonal PLB antibody showed that cAMP or Ca2+/calmodulin-dependent protein kinases phosphorylated PLB in an additive fashion. Inhibition of PP1 by inhibitor 1, CyA and TT maintained PLB in its phosphorylated state in a dose-dependent manner. The results of this study in which functional and biochemical experiments in cardiac SF were combined demonstrate that strong correlation exists between the phosphorylation-dephosphorylation cycle of PLB and calcium uptake.

Effects of protein phosphatase and kinase inhibitors on Ca2+ and Cl- currents in guinea pig ventricular myocytes.

It is well-established that in heart, both the L-type Ca2+ channel and the cystic fibrosis transmembrane conductance regulator Cl- channel are regulated by cAMP-dependent phosphorylation. However, it is not clear whether both of these channels are regulated in concert by protein kinase A (PKA) or whether there are mechanisms that independently control the phosphorylation of these two PKA targets. The purpose of this study was to compare the effects of various protein phosphatase and protein kinase inhibitors on these two ionic currents (ICa and ICl) in guinea pig ventricular myocytes to gain insight into these questions. We found that both the stimulation and washout of the effects of isoproterenol on ICl are about twice as fast as the effects on ICa, probably because the dephosphorylation reaction for ICl is faster than that for ICa. In contrast, inhibition of protein phosphatases with 10 microM microcystin stimulated both ICa and ICl, but the stimulation of ICl was much slower and smaller than the stimulation of ICa. The effect of microcystin was inhibited by staurosporine (Ki = 171.5 and 161 nM for ICa and ICl, respectively), suggesting that the stimulation was due to a kinase. The kinase was not protein kinase C (PKC) because it was not inhibited by the specific pseudosubstrate inhibitor of PKC, PKC(19-31), and it was not PKA because it was not inhibited by adenosine 3',5'-cyclic phosphorothioate. These results suggest that although both the Ca2+ and Cl- channels are regulated by cAMP-dependent phosphorylation, another protein kinase may also regulate these channels, and the kinetics of the response of the channels to phosphorylation can be modulated independently by protein phosphatases.

P-78 - Effects of protein phosphatases on the activation of guinea pig neutrophils.

P-78 - Effects of protein phosphatases on the activation of guinea pig neutrophils.

Effects of protein phosphatase and kinase inhibitors on the cardiac L-type Ca current suggest two sites are phosphorylated by protein kinase A and another protein kinase.

We previously showed (Frace, A.M. and H.C. Hartzell. 1993. Journal of Physiology. 472:305-326) that internal perfusion of frog atrial myocytes with the nonselective protein phosphatase inhibitors microcystin or okadaic acid produced an increase in the L-type Ca current (ICa) and a decrease in the delayed rectifier K current (IK). We hypothesized that microcystin revealed the activity of a protein kinase (PKX) that was basally active in the cardiac myocyte that could phosphorylate the Ca and K channels or regulators of the channels. The present studies were aimed at determining the nature of PKX and its phosphorylation target. The effect of internal perfusion with microcystin on ICa or IK was not attenuated by inhibitors of protein kinase A (PKA). However, the effect of microcystin on ICa was largely blocked by the nonselective protein kinase inhibitors staurosporine (10-30 nM), K252a (250 nM), and H-7 (10 microM). Staurosporine and H-7 also decreased the stimulation of ICa by isoproterenol, but K252a was more selective and blocked the ability of microcystin to stimulate ICa without significantly reducing isoproterenol-stimulated current. Internal perfusion with selective inhibitors of protein kinase C (PKC), including the autoinhibitory pseudosubstrate PKC peptide (PKC(19-31)) and a myristoylated derivative of this peptide had no effect. External application of several PKC inhibitors had negative side effects that prevented their use as selective PKC inhibitors. Nevertheless, we conclude that PKX is not PKC. PKA and PKX phosphorylate sites with different sensitivities to the phosphatase inhibitors calyculin A and microcystin. In contrast to the results with ICa, the effect of microcystin on IK was not blocked by any of the kinase inhibitors tested, suggesting that the effect of microcystin on IK may not be mediated by a protein kinase but may be due to a direct effect of microcystin on the IK channel.

Regulation of the cystic fibrosis transmembrane conductance regulator Cl- channel by specific protein kinases and protein phosphatases.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a regulated Cl- channel; in secretory epithelia, it is located in the apical membrane where it regulates transepithelial Cl- secretion. Previous studies have shown that cAMP-dependent protein kinase (PKA) can phosphorylate and activate CFTR Cl- channels. We asked whether other kinases would phosphorylate CFTR in vitro and activate CFTR Cl- channels in excised, inside-out patches of membrane from NIH 3T3 fibroblasts stably expressing recombinant CFTR. We found that both Ca(2+)-independent and Ca(2+)-dependent isoforms of protein kinase C (PKC) activated the CFTR Cl- channel. Consistent with this finding, PKC also phosphorylated CFTR in vitro. In contrast, the multifunctional Ca2+/calmodulin-dependent protein kinase failed to either activate or to phosphorylate CFTR Cl- channels, suggesting that this enzyme has no direct effect on CFTR. We found that cGMP-dependent protein kinase (cGK) (purified from bovine lung) phosphorylated CFTR in vitro. However, cGMP failed to increase the apical membrane Cl- permeability in human airway epithelia, and addition of cGMP, ATP, and cGK failed to activate CFTR Cl- channels. These results suggest that if cGK phosphorylates CFTR in vivo, it does so at sites not involved in CFTR Cl- channel activation. Because cAMP-dependent activation of CFTR Cl- channels and Cl- secretion in intact cells is reversible, we asked whether specific phosphatases can dephosphorylate and inactivate CFTR Cl- channels. Addition of protein phosphatase 2A (PP2A) decreased PKA-activated current by 67% within 10 min. The phosphatase inhibitor calyculin-A blocked the effect of PP2A. In contrast, neither protein phosphatases 1, 2B, nor two preparations of alkaline phosphatase inactivated PKA-phosphorylated CFTR Cl- channels. The effects of protein phosphatases on CFTR function were paralleled by their ability to dephosphorylate CFTR in vitro. Our data indicate that CFTR Cl- channels can be phosphorylated and activated by PKA as well as by Ca(2+)-dependent and Ca(2+)-independent isoforms of PKC and can be dephosphorylated and thus inactivated by PP2A.

Regulation of norepinephrine secretion in permeabilized PC12 cells by Ca2(+)-stimulated phosphorylation. Effects of protein phosphatases and phosphatase inhibitors.

Protein phosphatases and phosphatase inhibitors were used to examine the role of protein phosphorylation in the regulation of norepinephrine secretion in digitonin-permeabilized PC12 cells. The addition of an exogenous type 2A protein phosphatase caused as much as a 70% decrease in Ca2(+)-dependent norepinephrine secretion. In the presence of okadaic acid, a potent inhibitor of type 2A protein phosphatases, phosphatase 2A had no effect on secretion. The addition of exogenous calcineurin, a Ca2(+)-calmodulin-stimulated phosphatase, also caused decrease in Ca2(+)-dependent secretion, but on a molar basis it was less effective than phosphatase 2A. Two phosphatase inhibitors, 1-naphthylphosphate and sodium pyrophosphate, caused 75-100% increases in the amount of norepinephrine secreted in the absence of Ca2+ without affecting the amount of norepinephrine secreted in the presence of Ca2+. This stimulation of Ca2(+)-independent secretion by 1-naphthylphosphate and pyrophosphate suggests that there is a slow rate of Ca2(+)-independent phosphorylation and that phosphorylation triggers secretion. Unlike the results obtained in the presence of ATP, secretion in the presence of adenosine-5'-O-(3-thiotriphosphate), ATP gamma S, was not affected by the addition of type 2A protein phosphatase or by the addition of phosphatase inhibitors. These results are consistent with secretion in these permeabilized cells being regulated by a Ca2(+)-stimulated phosphorylation.