Increase In Protein Phosphatase Activity Research Articles

NADPH oxidase 1 supports proliferation of colon cancer cells by modulating reactive oxygen species-dependent signal transduction

Reactive oxygen species (ROS) play a critical role in cell signaling and proliferation. NADPH oxidase 1 (NOX1), a membrane-bound flavin dehydrogenase that generates O2˙̄, is highly expressed in colon cancer. To investigate the role that NOX1 plays in colon cancer growth, we used shRNA to decrease NOX1 expression stably in HT-29 human colon cancer cells. The 80–90% decrease in NOX1 expression achieved by RNAi produced a significant decline in ROS production and a G1/S block that translated into a 2–3-fold increase in tumor cell doubling time without increased apoptosis. The block at the G1/S checkpoint was associated with a significant decrease in cyclin D1 expression and profound inhibition of mitogen-activated protein kinase (MAPK) signaling. Decreased steady-state MAPK phosphorylation occurred concomitant with a significant increase in protein phosphatase activity for two colon cancer cell lines in which NOX1 expression was knocked down by RNAi. Diminished NOX1 expression also contributed to decreased growth, blood vessel density, and VEGF and hypoxia-inducible factor 1α (HIF-1α) expression in HT-29 xenografts initiated from NOX1 knockdown cells. Microarray analysis, supplemented by real-time PCR and Western blotting, revealed that the expression of critical regulators of cell proliferation and angiogenesis, including c-MYC, c-MYB, and VEGF, were down-regulated in association with a decline in hypoxic HIF-1α protein expression downstream of silenced NOX1 in both colon cancer cell lines and xenografts. These studies suggest a role for NOX1 in maintaining the proliferative phenotype of some colon cancers and the potential of NOX1 as a therapeutic target in this disease.

Tu1900 Indigo Naturalis and Indigo Ameliorate Murine Dextran Sodium Sulfate-Induced Colitis Through the Activation of Aryl Hydrocarbon Receptor

L-type Ca2+ channel activity was assayed in L6 cells as the rate of nifedipine-sensitive Ba2+ influx in a depolarizing medium. In the absence of extracellular Ca2+, activation of protein kinase C (PKC) with phorbol-12-myristate-13-acetate (PMA) or thymeleatoxin (TMX) inhibited Ba2+ influx by 38%. Thapsigargin (Tg), a selective inhibitor of the Ca2+-ATPase in the sarcoplasmic reticulum, evoked a rise in the cytosolic Ca2+ concentration ([Ca2+]i) in a Ca2+-free medium from 30 to ∼80 nM. This [Ca2+]i increase declined slowly, giving rise to a modest elevation of [Ca2+]i that persisted for >5 min. The inhibitory effects of PMA and TMX on channel activity were abolished when tested in Tg-treated cells in a Ca2+-free medium. However, when the Ca2+ ionophore, ionomycin, was applied with Tg, PMA and TMX retained their inhibitory effect on L-type Ca2+ channel activity, suggesting that a lower amplitude and prolonged release of Ca2+ stores is necessary for abrogating PKC-mediated inhibition of LCC. Cyclosporin A (5 μM) and ascomycin (5 μM), inhibitors of the Ca2+/calmodulin-dependent protein phosphatase, calcineurin, fully restored the inhibitory effect of PMA and TMX on channel activity. Addition of 1 mM CaCl2 to the Tg-treated cells increased [Ca2+]i to ∼165 nM and also restored the inhibitory effects of PMA and TMX. These results indicate that a small, relatively prolonged [Ca2+]i increase elicited by passive depletion of internal Ca2+ stores led to activation of calcineurin, giving rise to an increase in protein phosphatase activity that counteracted the inhibitory effects of PKC on channel activity. A larger increase in [Ca2+]i via store-dependent Ca2+ entry enhanced the activity of PKC sufficiently to overcome the protein phosphatase activity of calcineurin. This study is the first to demonstrate that the regulation of L-type Ca2+ channels in a myocyte model involves a balance between the differential Ca2+ sensitivities and opposing actions of PKC and calcineurin.

Decreased Activity of the Na + /H + Exchanger by Phosphodiesterase 5A Inhibition Is Attributed to an Increase in Protein Phosphatase Activity

The beneficial effect of phosphodiesterase 5A inhibition in ischemia/reperfusion injury and cardiac hypertrophy is well established. Inhibition of the cardiac Na(+)/H(+) exchanger (NHE-1) exerts beneficial effects on these same conditions, and a possible link between these therapeutic strategies was suggested. Experiments were performed in isolated cat cardiomyocytes to gain insight into the intracellular pathway involved in the reduction of NHE-1 activity by phosphodiesterase 5A inhibition. NHE-1 activity was assessed by the rate of intracellular pH recovery from a sustained acidic load in the absence of bicarbonate. Phosphodiesterase 5A inhibition with sildenafil (1 μmol/L) did not affect basal intracellular pH; yet, it did decrease proton efflux (J(H); in millimoles per liter per minute) after the acidic load (proton efflux: 6.97±0.43 in control versus 3.31±0.58 with sildenafil; P<0.05). The blockade of both protein phosphatase 1 and 2A with 100 nmol/L of okadaic acid reverted the sildenafil effect (proton efflux: 6.77±0.82). In contrast, selective inhibition of protein phosphatase 2A (1 nmol/L of okadaic acid or 100 μmol/L of endothall) did not (3.86±1.0 and 2.61±1.2), suggesting that only protein phosphatase 1 was involved in sildenafil-induced NHE-1 inhibition. Moreover, sildenafil prevented the acidosis-induced increase in NHE-1 phosphorylation without affecting activation of the extracellular signal-regulated kinase 1/2-p90(RSK) pathway. Our results suggest that phosphodiesterase 5A inhibition decreases NHE-1 activity, during intracellular pH recovery after an acidic load, by a protein phosphatase 1-dependent reduction in NHE-1 phosphorylation.

Calcineurin activation by slow calcium release from intracellular stores suppresses protein kinase C regulation of L-type calcium channels in L6 cells

L-type Ca 2+ channel activity was assayed in L6 cells as the rate of nifedipine-sensitive Ba 2+ influx in a depolarizing medium. In the absence of extracellular Ca 2+, activation of protein kinase C (PKC) with phorbol-12-myristate-13-acetate (PMA) or thymeleatoxin (TMX) inhibited Ba 2+ influx by 38%. Thapsigargin (Tg), a selective inhibitor of the Ca 2+-ATPase in the sarcoplasmic reticulum, evoked a rise in the cytosolic Ca 2+ concentration ([Ca 2+] i) in a Ca 2+-free medium from 30 to ∼80 nM. This [Ca 2+] i increase declined slowly, giving rise to a modest elevation of [Ca 2+] i that persisted for >5 min. The inhibitory effects of PMA and TMX on channel activity were abolished when tested in Tg-treated cells in a Ca 2+-free medium. However, when the Ca 2+ ionophore, ionomycin, was applied with Tg, PMA and TMX retained their inhibitory effect on L-type Ca 2+ channel activity, suggesting that a lower amplitude and prolonged release of Ca 2+ stores is necessary for abrogating PKC-mediated inhibition of LCC. Cyclosporin A (5 μM) and ascomycin (5 μM), inhibitors of the Ca 2+/calmodulin-dependent protein phosphatase, calcineurin, fully restored the inhibitory effect of PMA and TMX on channel activity. Addition of 1 mM CaCl 2 to the Tg-treated cells increased [Ca 2+] i to ∼165 nM and also restored the inhibitory effects of PMA and TMX. These results indicate that a small, relatively prolonged [Ca 2+] i increase elicited by passive depletion of internal Ca 2+ stores led to activation of calcineurin, giving rise to an increase in protein phosphatase activity that counteracted the inhibitory effects of PKC on channel activity. A larger increase in [Ca 2+] i via store-dependent Ca 2+ entry enhanced the activity of PKC sufficiently to overcome the protein phosphatase activity of calcineurin. This study is the first to demonstrate that the regulation of L-type Ca 2+ channels in a myocyte model involves a balance between the differential Ca 2+ sensitivities and opposing actions of PKC and calcineurin.

Peroxynitrite Increases Protein Phosphatase Activity and Promotes the Interaction of Phospholamban with Protein Phosphatase 2a in the Myocardium

Nitric oxide and superoxide react to form the potent oxidant peroxynitrite. The production of peroxynitrite increases during the pathogenesis of heart failure and is detrimental to myocardial function. We previously demonstrated that high levels of peroxynitrite decrease myocardial contraction by reducing phospholamban (PLB) phosphorylation through a protein phosphatase-dependent mechanism. However, we did not examine the direct effect of peroxynitrite on protein phosphatase activity in the myocardium or the specific protein phosphatase which is activated. Here we test: 1.) the effect of SIN-1 (peroxynitrite donor) on protein phosphatase activity in whole heart homogenates using a colorimetric assay, and 2.) the effect of SIN-1 on the interaction of PLB with protein phosphatase 1 (PP1) and protein phosphatase 2a (PP2a) using co-immunoprecipitation. SIN-1 induced a 63% increase in total protein phosphatase activity (1.6±0.2 vs. 2.6±0.3 nmol/min/mg, p<0.05 vs. Control), which was abolished with specific PP1/PP2a inhibition using okadaic acid (1.4±0.2 nmol/min/mg, p<0.05 vs. SIN-1). Since okadaic acid prevented the effects of SIN-1, we next examined the effect of SIN-1 on the interaction of PLB with PP1 and PP2a. SIN-1 increased the interaction of PLB with PP2a by 350% (0.6± 0.3 vs. 2.7±0.7 A.U., p<0.05 vs. Control), but had no effect on the interaction with PP1. The peroxynitrite scavenger, urate, prevented both the SIN-1-induced increase in protein phosphatase activity and the interaction of PLB with PP2a, thus implicating peroxynitrite as the causal species. The results of this study provide further insight into the mechanism through which high levels of peroxynitrite serve to decrease PLB phosphorylation and myocardial contraction. Therefore, increased peroxynitrite production may play a key role in heart failure where protein phosphatase activity is increased and PLB phosphorylation is decreased, ultimately leading to contractile dysfunction.

Peroxynitrite increases protein phosphatase activity and promotes the interaction of phospholamban with protein phosphatase 2a in the myocardium

High levels of peroxynitrite have been shown to decrease cardiomyocyte contraction through a reduction in phospholamban (PLB) phosphorylation. However, previous reports did not examine the direct effect of peroxynitrite on protein phosphatase activity in the myocardium or the role of specific phosphatases. Here we test the effect of the peroxynitrite donor SIN-1 on protein phosphatase activity in whole heart homogenates, as well as the interaction of PLB with protein phosphatase 1 (PP1) and 2a (PP2a). SIN-1 (200 μmol/L) induced a significant increase in protein phosphatase activity, which was alleviated with the specific PP1/PP2a inhibitor okadaic acid. Conversely, lower concentrations of SIN-1 and the nitric oxide donor spermine NONOate (300 μmol/L) were both without effect on phosphatase activity. We next examined the effect of SIN-1 on the interaction of PLB with PP1 and PP2a using co-immunoprecipitation, since okadaic acid inhibited the effects of SIN-1 in our current and previous studies. SIN-1 significantly increased the interaction of PLB with PP2a, but had no effect on the interaction between PLB and PP1. Urate, a peroxynitrite scavenger, inhibited the effects of SIN-1 on phosphatase activity and the interaction of PLB with PP2a, thus implicating peroxynitrite as the causal species. The results of this study provide further insight into the mechanism through which high levels of peroxynitrite serve to decrease PLB phosphorylation and myocardial contraction. Therefore, peroxynitrite signaling could play a key role in the contractile dysfunction manifested in heart failure where peroxynitrite production and protein phosphatase activity are increased and PLB phosphorylation is decreased.

Protein phosphatase 1 activation and alternative splicing of Bcl‐X and Mcl‐1 by EGCG + ibuprofen

Epigallocatechin-3-gallate (EGCG) and ibuprofen synergistically act to suppress proliferation and enhance apoptosis of prostate cancer cell lines, PC-3 and LNCaP. The purpose of this study was to investigate the mechanism of underlying this synergism. Most interestingly, EGCG + ibuprofen treatment in PC-3 cells resulted in altering the ratio of the splice variants of Bcl-X and Mcl-1, downregulating the mRNA levels of anti-apoptotic Bcl-X(L) and Mcl-1(L) with a concomitant increase in the mRNA levels of pro-apoptotic Bcl-X(s) and Mcl-1(s). However, there were no apparent changes in splicing variants in either ibuprofen or EGCG treated cells. Induction of alternative splicing was correlated with increased activity of protein phosphatase 1 (PP1) in EGCG + ibuprofen-treated cells, since pretreatment with calyculin A and tautomycin blocked EGCG + ibuprofen-induced alternative splicing in PC-3 cells in contrast to pretreatment with okadaic acid. On the other hand, EGCG + ibuprofen treatment in LNCaP cells did not alter splicing variants of Bcl-X and Mcl-1, despite the increase in protein phosphatase activity. In both cell lines, EGCG + ibuprofen inhibited cell proliferation synergistically. Taken together, this study demonstrate for the first time that EGCG + ibuprofen upregulated PP1 activity, which in turn induced alternative splicing of Bcl-X and Mcl-1 in a cell-type specific manner. Our study also demonstrates that the activation of PP1 contributes to the alternative splicing of Mcl-1.

Suppressive effect of regucalcin on protein phosphatase activity in the heart cytosol of normal and regucalcin transgenic rats

The role of regucalcin, a regulatory protein in intracellular signaling pathway, in the regulation of protein phosphatase activity in the heart muscle cytosol was investigated by using normal (wild-type) and regucalcin transgenic (TG) rats. Protein phosphatase activity was assayed in a reaction mixture containing the cytosolic protein in the presence of phosphotyrosine, phosphoserine, and phosphothreonine. The addition of calcium chloride (10 and 20 microM) in the enzyme reaction mixture caused a significant increase in protein phosphatase activity toward three phosphoaminoacids. Trifluoperazine (10 and 20 microM), an antagonist of calmodulin, completely inhibited calcium (10 microM) addition-increased protein phosphatase activity toward three phosphoaminoacids. Moreover, the calcium (10 microM)-increased enzyme activity toward phosphoserine and phosphothreonine was significantly enhanced by the addition of calmodulin (2.5 or 5 microg/ml). Such an enhancement was not seen in the presence of phosphotyrosine. Regucalcin (10(-9) and 10(-8) M) significantly inhibited protein phosphatase activity toward three phosphoaminoacids in the presence of ethylene glycol bis (2-aminoethlether) N,N,N',N'-tetraacetic acid (EGTA; 1 mM), without Ca2+ addition. The inhibitory effect of regucalcin (10(-10)-10(-8) M) was also seen in the presence of calcium chloride (10 microM). Western blot analysis showed a remarkable expression of regucalcin protein in the cytosol of heart of regucalcin TG female rats as compared with that of wild-type female rats. Protein phosphatase activity toward three phosphoaminoacids was significantly decreased in the heart cytosol of TG rats. The enhancing effect of calcium (10 microM) addition on protein phosphatase activity toward three phosphoaminoacids was not seen in the heart cytosol of TG rats. This study demonstrates that endogenous regucalcin plays a suppressive role in the regulation of protein phosphatase activity in rat heart cytoplasm.

Elevated levels of protein phosphatase 1 and phosphatase 2A may contribute to cardiac dysfunction in diabetes

Although protein phosphorylation and dephosphorylation are known to regulate the activities of different enzymes, sufficient information on the role of dephosphorylation in cardiac function is not available. Since protein phosphatases mediate dephosphorylation, it is possible that cardiac dysfunction induced by diabetes may be due to alterations in the activities of these enzymes. We therefore determined cardiac protein phosphatase activity as well as protein contents of phosphatase 1 and phosphatase 2A in diabetic animals. For this purpose, rats were made diabetic by administering a single intravenous injection of streptozotocin (65 mg/kg body weight) and hearts were examined after 1, 2, 3, 4 and 8 weeks. Some of the 4-week diabetic animals received subcutaneous injections of insulin (3 U/day) for a further period of 4 weeks. Cardiac dysfunction was evident after 2 weeks of inducing diabetes and deteriorated further with time. A significant increase in protein phosphatase activity appeared after 1 week and persisted until 8 weeks. Increased protein phosphatase activity in the diabetic heart was associated with a corresponding increase in the protein contents of both phosphatase 1 and phosphatase 2A. Insulin treatment partly prevented the changes observed in diabetic animals. The results suggest that increased protein phosphatase activities and subsequent enhanced protein dephosphorylation may play a role in diabetes-induced cardiac dysfunction.

Acute glucose starvation activates the nuclear localization signal of a stress-specific yeast transcription factor.

In yeast, environmental conditions control the transcription factor Msn2, the nuclear accumulation and function of which serve as a sensitive indicator of nutrient availablity and environmental stress load. We show here that the nuclear localization signal (NLS) of Msn2 is a direct target of cAMP-dependent protein kinase (cAPK). Genetic analysis suggests that Msn2-NLS function is inhibited by phosphorylation and activated by dephosphorylation. Msn2-NLS function is unaffected by many stress conditions that normally induce nuclear accumulation of full-length Msn2. The Msn2-NLS phosphorylation status is, however, highly sensitive to carbohydrate fluctuations during fermentative growth. Dephosphorylation occurs in >2 min after glucose withdrawal but the effect is reversed rapidly by refeeding with glucose. This response to glucose depletion is due to changes in cAPK activity rather than an increase in protein phosphatase activity. Surprisingly, the classical glucose-sensing systems are not connected to this rapid response system. Our results further imply that generic stress signals do not cause short-term depressions in cAPK activity. They operate on Msn2 by affecting an Msn5-dependent nuclear export and/or retention mechanism.

Endogenous regucalcin suppresses the enhancement of protein phosphatase activity in the cytosol and nucleus of kidney cortex in calcium-administered rats.

The suppressive role of endogenous regucalcin (RC), which is a regulatory protein of calcium signaling, in the enhancement of protein phosphatase activity (PPA) in the cytosol and nucleus of kidney cortex in calcium-administered rats was investigated. Calcium content in the kidney cortex was significantly increased at 0.5-5 h after a single intraperitoneal administration of calcium chloride solution (10 mg Ca/100 g body weight) to rats. The analysis with Western blotting of RC protein showed that RC levels in the cytosol and nucleus were significantly increased 0.5-5 h after the administration of calcium (10 mg/100 g). PPA toward phosphotyrosine, phosphoserine, and phosphothreonine was found in the cytosol and nucleus of kidney cortex. PPA toward three phosphoamino acids in the cytosol and nucleus was significantly increased by the administration of calcium (10 mg/100 g). The presence of anti-RC monoclonal antibody (25 ng/ml) in the enzyme reaction caused a significant increase in PPA toward phosphotyrosine, phosphoserine, and phosphothreonine in the cytosol and nucleus of kidney cortex in normal rats. The effect of anti-RC monoclonal antibody (25 ng/ml) in increasing PPA toward three phosphoamino acids in the cytosol and nucleus was significantly enhanced in calcium-administered rats. The effect of anti-RC monoclonal antibody (25 ng/ml) in increasing PPA in the cytosol and nucleus of normal rats and calcium-administered rats was completely abolished by the addition of RC (10(- 6) M) in the enzyme reaction mixture. The present study suggests that endogenous RC suppresses the enhancement of PPA in the cytosol and nucleus of kidney cortex in calcium-administered rats.

Protein Phosphatase-Mediated Regulation of Protein Kinase C during Long-Term Depression in the Adult HippocampusIn Vivo

The neural substrates of learning and memory are thought to involve use-dependent long-term changes in synaptic function, including long-term depression (LTD) of synaptic strength. One biochemical event hypothesized to contribute to the maintenance and expression of LTD is decreased protein phosphorylation, caused by a decrease in protein kinase activity and/or an increase in protein phosphatase activity. We tested whether the activity of protein kinase C (PKC) decreases after the induction of LTD in area CA1 of the adult hippocampus in vivo, and then investigated the mechanism responsible for the LTD-associated alteration in PKC activity. We found that LTD was associated with a significant decrease in both autonomous and cofactor-dependent PKC activity. The decrease in PKC activity was prevented by NMDA receptor blockade and was not accompanied by a decrease in the level of either PKCalpha, beta, gamma, or zeta. Western blot analysis with phosphospecific antibodies revealed that phosphorylation of Ser-657 on the catalytic domain of PKCalpha (Ser-660 on PKCbetaII) was decreased significantly after the induction of LTD, and that this dephosphorylation was prevented by the protein phosphatase inhibitor okadaic acid. The decrease in autonomous and cofactor-dependent PKC activity likewise was prevented by okadaic acid. These findings suggest that LTD in the adult hippocampus in vivo involves a decrease in PKC activity that is mediated, at least in part, by dephosphorylation of the catalytic domain of PKC by protein phosphatases activated after LTD-inducing stimulation. Our findings are consistent with the idea that protein dephosphorylation contributes to the expression of LTD.

Expression of Ca 2+-binding protein regucalcin in rat brain neurons: inhibitory effect on protein phosphatase activity

The expression of Ca 2+-binding protein regucalcin and its role in the regulation of protein phosphatase activity in rat brain neuronal cells obtained with primary culture was investigated. The expression of regucalcin mRNA was demonstrated by reverse transcription-polymerase chain reaction (RT-PCR) analysis in brain neuronal cells using rat regucalcin-specific primers. Moreover, regucalcin protein in brain neuronal cells was detected by Western blot analysis using a polyclonal rabbit anti-regucalcin antibody. The presence of anti-regucalcin monoclonal antibody (20 or 50 ng/ml) in the enzyme reaction mixture caused a significant increase in protein phosphatase activity toward phosphotyrosine, phosphoserine and phosphothreonine in the reaction mixture containing the cytosol of neuronal cell homogenates. This increase was completely prevented by the addition of regucalcin (10 −8 M). Protein phosphatase activity toward three phosphoaminoacids was significantly elevated by the addition of Ca 2+ (100 μM) and calmodulin (5 μg/ml). This elevation was completely blocked by the addition of regucalcin (10 −8 M). The present study demonstrates that regucalcin is expressed in rat brain neuronal cells, and that it has an inhibitory effect on protein phosphatase activity in the cells.

Control of cholecystokinin receptor dephosphorylation in pancreatic acinar cells

Phosphorylation of cell surface receptors regulates the physiological response to many hormones and neurotransmitters. We have demonstrated that the receptor for cholecystokinin (CCK), the major secretagogue for the exocrine pancreas, is phosphorylated on serines in response to CCK (Klueppelberg, U.G., Gates, L.K., Gorelick, F.S., and Miller, L.J. (1991) J. Biol. Chem. 266, 2403-2408). Receptor phosphorylation was transient, even in the continued presence of agonist, and suggested a role for pancreatic protein phosphatases (PP) in regulating receptor phosphorylation in the intact cell. Treatment of acinar cells with okadaic acid increased the extent and duration of receptor phosphorylation induced by CCK. Receptor phosphorylated in the intact cell in response to CCK was used as substrate to analyze protein phosphatases in pancreatic acinar cell extracts. The predominant CCK receptor phosphatase activity was found in the cytosol and was potently inhibited by okadaic acid (IC50 = 0.2 nM). This phosphatase activity was unaffected by inhibitor-2, Ca2+, or Mg2+, suggesting that the major receptor phosphatase was PP-2A. Stimulation of cells with CCK resulted in a 3-fold increase in protein phosphatase activity toward the CCK receptor, at the same time that phosphorylase a phosphatase activity was unchanged. This increase in receptor phosphatase activity was reflected only in the cytosol, with the particulate activity unchanged. Consistent with this subcellular localization, the hormone-regulated receptor phosphatase activity was PP-2A, with chromatographic separation demonstrating activity in both PP-2A1 and PP-2A2 forms. These data suggest that CCK coordinates the activity of both protein kinases and phosphatases acting on the CCK receptor to control the extent and duration of receptor phosphorylation in the pancreatic acinar cell.

Aldosterone-induced increase in protein phosphatase activity of toad bladder.

A study has been carried out of the effect of aldosterone on the endogenous phosphorylation and dephosphorylation of membrane-bound and of soluble proteins from toad bladder. Membrane-bound protein D (apparent molecular weight, 49,000), a protein which may possibly be involved in the regulation of sodium transport across the mucosal epithelium of toad bladder, contained a substantial fraction of the radioactive phosphate incorporated into membrane proteins; moreover, it was the only protein to appear consistently in autoradiographs of polyacrylamide gels of phosphorylated membrane proteins. Pretreatment of toad bladder slices with aldosterone caused an increase in the endogenous dephosphorylation of membrane-bound protein D. A half-maximal increase in this dephosphorylation occurred at an aldosterone concentration of 20-40 nM. The increase in protein D phosphatase activity induced by aldosterone was prevented by inhibitors of RNA and protein synthesis as well as by spironolactone, a specific antagonist of aldosterone. The mineralocorticoid, 9alpha-fluorohydrocortisone, also increased protein D phosphatase activity, but testosterone did not. Aldosterone also increased the removal of [(32)P]phosphate from protein D in the cell sap. In contrast to the increase in protein D phosphatase activity, aldosterone had little effect on the phosphorylation of protein D by endogenous protein D kinase. In some experiments, effects of aldosterone and of cAMP, qualitatively similar to those found with protein D, were also observed on the phosphorylation and dephosphorylation of a protein with an apparent molecular weight of 37,000, in both the microsomal and cell sap fractions. No consistent effect of preincubation with aldosterone or of cAMP was observed on any membrane-bound or cell sap protein other than protein D and the 37,000 dalton protein.