Protein kinase C activation antagonizes melatonin-induced pigment aggregation in Xenopus laevis melanophores

The pineal hormone, melatonin (5-methoxy N-acetyltryptamine) induces a rapid aggregation of melanin-containing pigment granules in isolated melanophores of Xenopus laevis. Treatment of melanophores with activators of protein kinase C (PKC), including phorbol esters, mezerein and a synthetic diacylglycerol, did not affect pigment granule distribution but did prevent and reverse melatonin-induced pigment aggregation. This effect was blocked by an inhibitor of PKC, Ro 31-8220. The inhibitory effect was not a direct effect on melatonin receptors, per se, as the slow aggregation induced by a high concentration of an inhibitor of cyclic AMP-dependent protein kinase (PKA), adenosine 3',5'-cyclic monophosphothioate, Rp-diastereomer (Rp-cAMPS), was also reversed by PKC activation. Presumably activation of PKC, like PKA activation, stimulates the intracellular machinery involved in the centrifugal translocation of pigment granules along microtubules. alpha-Melanocyte stimulating hormone (alpha-MSH), like PKC activators, overcame melatonin-induced aggregation but this response was not blocked by the PKC inhibitor, Ro 31-8220. This data indicates that centrifugal translocation (dispersion) of pigment granules in Xenopus melanophores can be triggered by activation of either PKA, as occurs after alpha-MSH treatment, or PKC. The very slow aggregation in response to inhibition of PKA with high concentrations of Rp-cAMPS, suggests that the rapid aggregation in response to melatonin may involve multiple intracellular signals in addition to the documented Gi-mediated inhibition of adenylate cyclase.     

Rab32 regulates melanosome transport in Xenopus melanophores by protein kinase a recruitment

Intracellular transport is essential for cytoplasm organization, but mechanisms regulating transport are mostly unknown. In Xenopus melanophores, melanosome transport is regulated by cAMP-dependent protein kinase A (PKA). Melanosome aggregation is triggered by melatonin, whereas dispersion is induced by melanocyte-stimulating hormone (MSH). The action of hormones is mediated by cAMP: High cAMP in MSH-treated cells stimulates PKA, whereas low cAMP in melatonin-treated cells inhibits it. PKA activity is typically restricted to specific cell compartments by A-kinase anchoring proteins (AKAPs). Recently, Rab32 has been implicated in protein trafficking to melanosomes and shown to function as an AKAP on mitochondria. Here, we tested the hypothesis that Rab32 is involved in regulation of melanosome transport by PKA. We demonstrated that Rab32 is localized to the surface of melanosomes in a GTP-dependent manner and binds to the regulatory subunit RIIalpha of PKA. Both RIIalpha and Cbeta subunits of PKA are required for transport regulation and are recruited to melanosomes by Rab32. Overexpression of wild-type Rab32, but not mutants unable to bind PKA or melanosomes, inhibits melanosome aggregation by melatonin. Therefore, in melanophores, Rab32 is a melanosome-specific AKAP that is essential for regulation of melanosome transport.     

The protein kinase A-anchoring protein moesin is bound to pigment granules in melanophores

Major signaling cascades have been shown to play a role in the regulation of intracellular transport of organelles. In Xenopus melanophores, aggregation and dispersion of pigment granules are regulated by the second messenger cyclic AMP through the protein kinase A (PKA) signaling pathway. PKA is bound to pigment granules where it forms complexes with molecular motors involved in pigment transport. Association of PKA with pigment granules occurs through binding to A-kinase-anchoring proteins (AKAPs), whose identity remains largely unknown. In this study, we used mass spectrometry to examine an 80 kDa AKAP detected in preparations of purified pigment granules. We found that tryptic digests of granule protein fractions enriched in the 80 kDa AKAP contained peptides that corresponded to the actin-binding protein moesin, which has been shown to function as an AKAP in mammalian cells. We also found that recombinant Xenopus moesin interacted with PKA in vitro , copurified with pigment granules and bound to pigment granules in cells. Overexpression in melanophores of a mutant moesin lacking conserved PKA-binding domain did not affect aggregation of pigment granules but partially inhibited their dispersion. We conclude that Xenopus moesin is an AKAP whose PKA-scaffolding activity plays a role in the regulation of pigment dispersion in Xenopus melanophores.     

The protein-phosphatase inhibitor okadaic acid mimics MSH-induced and melatonin-reversible melanosome dispersion in Xenopus laevis melanophores.

The present study describes the ability of 315 nM okadaic acid to induce melanosome dispersion in cultured Xenopus laevis melanophores. This effect of okadaic acid is similar to that of a-melanocyte stimulating hormone (MSH) and can be reversed by melatonin treatment; it indicates that a member of the protein-phosphatase 1 or 2A families must be active for maintenance of the aggregated state. Higher concentrations of okadaic acid (1 microM) attenuate the response of Xenopus melanophores to melatonin leading to the hypothesis that melatonin action is mediated by the calcium/calmodulin activated phosphatase 2B. This hypothesis seems unlikely, however, since the calcium/calmodulin inhibitors TFP and W7 do not prevent melatonin-induced pigment aggregation, but instead induce aggregation on their own.     

Protein kinase A, which regulates intracellular transport, forms complexes with molecular motors on organelles

Major signaling cascades have been shown to play a role in the regulation of intracellular organelle transport . Aggregation and dispersion of pigment granules in melanophores are regulated by the second messenger cAMP through the protein kinase A (PKA) signaling pathway ; however, the exact mechanisms of this regulation are poorly understood. To study the role of signaling molecules in the regulation of pigment transport in melanophores, we have asked the question whether the components of the cAMP-signaling pathway are bound to pigment granules and whether they interact with molecular motors to regulate the granule movement throughout the cytoplasm. We found that purified pigment granules contain PKA and scaffolding proteins and that PKA associates with pigment granules in cells. Furthermore, we found that the PKA regulatory subunit forms two separate complexes, one with cytoplasmic dynein ("aggregation complex") and one with kinesin II and myosin V ("dispersion complex"), and that the removal of PKA from granules causes dissociation of dynein and disruption of dynein-dependent pigment aggregation. We conclude that cytoplasmic organelles contain protein complexes that include motor proteins and signaling molecules involved in different components of intracellular transport. We propose to call such complexes 'regulated motor units' (RMU).     

The Protein-Phosphatase Inhibitor Okadaic Acid Mimics MSH-Induced and Melatonin-Reversible Melanosome Dispersion in Xenopus laevis Melanophores

The present study describes the ability of 315 nM okadaic acid to induce melanosome dispersion in cultured Xenopus laevis melanophores. This effect of okadaic acid is similar to that of a-melanocyte stimulating hormone (MSH) and can be reversed by melatonin treatment; it indicates that a member of the protein-phosphatase 1 or 2A families must be active for maintenance of the aggregated state. Higher concentrations of okadaic acid (1 μM) attenuate the response of Xenopus melanophores to melatonin leading to the hypothesis that melatonin action is mediated by the calcium/calmodulin activated phosphatase 2B. This hypothesis seems unlikely, however, since the calcium/calmodulin inhibitors TFP and W7 do not prevent melatonin-induced pigment aggregation, but instead induce aggregation on their own.     

Regulation of melanosome movement by MAP kinase

Our objectives were to further characterize the signaling pathways in melatonin-induced aggregation in Xenopus melanophores, specifically to investigate a possible role of mitogen-activated protein kinase (MAPK). By Western blotting we found that melatonin activates MAPK, which precedes melanosome aggregation measured in a microplate reader. Activation of MAPK, tyrosine phosphorylation of a previously described 280-kDa protein, and melanosome aggregation are sensitive to PD98059, a selective inhibitor of MAPK kinase. The MAPK activation is also decreased by the adenylate cyclase stimulant forskolin. In summary, we found that MAPK is activated during melatonin-induced melanosome aggregation. Activation was decreased by an inhibitor of MAPK kinase, and by forskolin. In addition to inhibition of cyclic adenosine 3',5'-monophosphate (cAMP), reduction in protein kinase A activity (PKA), and activation of protein phosphatase 2A, we suggest that melatonin receptors activate the MAPK cascade and tyrosine phosphorylation of the 280-kDa protein. Although the cAMP/PKA signaling pathway is the most prominent, our data suggest that simultaneous activation of the MAPK cascade is of importance to obtain a completely aggregated state. This new regulatory mechanism of organelle transport by the MAPK cascade might be important in other eukaryotic cells.     

Phosphorylation of SET Protein at Ser171 by Protein Kinase D2 Diminishes Its Inhibitory Effect on Protein Phosphatase 2A

We previously reported that protein kinase D2 (PKD2) in T cells is promptly activated after T-cell receptor (TCR) stimulation and involved in the activation of interleukin-2 promoter and T cell death, and that one of its candidate substrate is SET protein, a natural inhibitor for protein phosphatase 2A (PP2A). In this study, we investigated the target amino acid residues of SET phosphorylated by PKD2 and the effects of phosphorylation of SET on PP2A phosphatase activity. In vitro kinase assay using various recombinant SET mutants having Ser/Thr to Ala substitutions revealed that Ser171 of SET is one of the sites phosphorylated by PKD2. Recombinant SET with phosphorylation-mimic Ser171 to Glu substitution reduced its inhibitory effects on PP2A phosphatase activity compared with Ser171 to Ala substituted or wild-type SET. In addition, knockdown of PKD2 in Jurkat cells by RNAi or treatment of human CD4⁺ T cell clone with the PKD2 inhibitor Gö6976 resulted in reduced PP2A activity after TCR-stimulation judged from phosphorylation status of Tyr307 of the catalytic subunit of PP2A. These results suggest that PKD2 is involved in the regulation of PP2A activity in activated T cells through phosphorylation of Ser171 of SET.     

Stimulation of Cell Elongation in Teleost Rod Photoreceptors by Distinct Protein Kinase Inhibitors

Abstract: The rod photoreceptors of teleost retinas elongate in the light. To characterize the role of protein kinases in elongation, pharmacological studies were carried out with rod fragments consisting of the motile inner segment and photosensory outer segment (RIS-ROS). Isolated RIS-ROS were cultured in the presence of membrane-permeant inhibitors that exhibit selective activity toward specific serine/threonine protein kinases. We report that three distinct classes of protein kinase inhibitors stimulated elongation in darkness: (1) cyclic AMP-dependent protein kinase (PKA)-selective inhibitors (H-89 and KT5720), (2) a protein kinase C (PKC)-selective inhibitor (GF 109203X) that affects most PKC isoforms, and (3) a kinase inhibitor (H-85) that does not affect PKC and PKA in vitro. Other kinase inhibitors tested neither stimulated elongation in darkness nor inhibited light-induced elongation; these include the myosin light chain kinase inhibitors ML-7 and ML-9, the calcium-calmodulin kinase II inhibitor KN-62, and inhibitors or activators of diacylglycerol-dependent PKCs (sphingosine, calphostin C, chelerythrine, and phorbol esters). The myosin light chain kinase inhibitors as well as the PKA and PKC inhibitors H-89 and GF 109203X all enhanced light-induced elongation. These observations suggest that light-induced RIS-ROS elongation is inhibited by both PKA and an unidentified kinase or kinases, possibly a diacylglycerol-independent form of PKC.     

L-NAME-induced dispersion of melanosomes in melanophores activates PKC, MEK and ERK1.

Melanosome movement represents a good model of cytoskeleton-mediated transport of organelles in eukaryotic cells. We recently observed that inhibiting nitric oxide synthase (NOS) with Nomega-nitro-L-arginine methyl ester (L-NAME) induced dispersion in melanophores pre-aggregated with melatonin. Activation of cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase (PKA) or calcium-dependent protein kinase (PKC) is known to cause dispersion. Also, PKC and NO have been shown to regulate the mitogen/extracellular signal-regulated kinase (MEK)-ERK pathway. Accordingly, our objective was to further characterize the signaling pathway of L-NAME-induced dispersion. We found that the dispersion was decreased by staurosporine and PD98059, which respectively inhibit PKC and MEK, but not by the PKA inhibitor H89. Furthermore, Western blotting revealed that ERK1 kinase was phosphorylated in L-NAME-dispersed melanophores. L-NAME also caused dispersion in latrunculin-B-treated cells, suggesting that this effect is not due to inhibition of the melatonin signaling pathway. Summarizing, we observed that PKC and MEK inhibitors decreased the L-NAME-induced dispersion, which caused phosphorylation of ERK1. Our results also suggest that NO is a negative regulator of phosphorylations that leads to organelle transport.     

Bidirectional pigment granule movements of melanophores are regulated by protein phosphorylation and dephosphorylation

Studies were conducted to investigate the molecular basis for bidirectional pigment granule transport in digitonin-lysed melanophores. Pigment granule dispersion, but not aggregation, required cAMP and resulted in the phosphorylation of a 57 kd polypeptide. cAMP-dependent protein kinase inhibitor prevented this phosphorylation as well as pigment dispersal. In contrast, both pigment aggregation and the concomitant dephosphorylation of the 57 kd polypeptide were blocked by phosphatase inhibitors. These data support a model in which pigment dispersion and aggregation require protein phosphorylation and dephosphorylation, respectively. Furthermore, studies using the ATP analog, ATP gamma S, suggest either that protein phosphorylation alone is sufficient for dispersion or that transport is mediated by a unique force-generating ATPase that can use ATP gamma S for hydrolyzable energy.     

l‐NAME‐Induced Dispersion of Melanosomes in Melanophores Activates PKC,MEK and ERK1

Melanosome movement represents a good model of cytoskeleton‐mediated transport of organelles in eukaryotic cells. We recently observed that inhibiting nitric oxide synthase (NOS) with Nω‐nitro‐l ‐arginine methyl ester (l ‐NAME) induced dispersion in melanophores pre‐aggregated with melatonin. Activation of cyclic adenosine 3′,5′‐monophosphate (cAMP)‐dependent protein kinase (PKA) or calcium‐dependent protein kinase (PKC) is known to cause dispersion. Also, PKC and NO have been shown to regulate the mitogen/extracellular signal‐regulated kinase (MEK)‐ERK pathway. Accordingly, our objective was to further characterize the signaling pathway of l ‐NAME‐induced dispersion. We found that the dispersion was decreased by staurosporine and PD98059, which respectively inhibit PKC and MEK, but not by the PKA inhibitor H89. Furthermore, Western blotting revealed that ERK1 kinase was phosphorylated in l ‐NAME‐dispersed melanophores. l ‐NAME also caused dispersion in latrunculin‐B‐treated cells, suggesting that this effect is not due to inhibition of the melatonin signaling pathway. Summarizing, we observed that PKC and MEK inhibitors decreased the l ‐NAME‐induced dispersion, which caused phosphorylation of ERK1. Our results also suggest that NO is a negative regulator of phosphorylations that leads to organelle transport.     

Regulation of alpha(1)-adrenoceptor-linked phosphoinositide metabolism in cultured glia: involvement of protein phosphatases and kinases

Noradrenaline-stimulated phosphoinositide breakdown in cultured glia was found to be mediated by alpha(1A)-adrenoceptors. The alpha(1A)-selective agonist A61603 was as effective as noradrenaline in eliciting 3H-inositol phosphate (IP) accumulation but was approximately 50-fold more potent. In addition, the use of selective antagonists revealed a clear rank order of potency in the ability of these drugs to reverse the effect of noradrenaline on phosphoinositide breakdown: RS17053 (alpha(1A)-selective) >AH11110A (alpha(1B)-selective)>BMY7378 (alpha(1D)-selective). Pre-treatment of cultured glia with the protein phosphatase inhibitor okadaic acid resulted in a concentration- and time-dependent reduction in noradrenaline-evoked 3H-IP accumulation. This effect was mimicked by, but was not additive with, a phorbol ester, was reversed by protein kinase C (PKC) inhibitors and was not evident in cells which had been PKC depleted. The ability of cell extracts to dephosphorylate radiolabelled glycogen phosphorylase revealed the presence of the phosphatases PP1 and PP2A in almost equal abundance. Okadaic acid pre-treatment of intact cultures elicited a marked reduction in total phosphatase activity, particularly that mediated by PP2A. We also determined the effect of okadaic acid pre-treatment on PKC and cyclic AMP-dependent protein kinase (PKA) activities in these cells. PKC and PKA activities in cell extracts were assessed by determining the incorporation of 32P into histone and kemptide, respectively. Okadaic acid elicited increases in both Ca(2+)-dependent and Ca(2+)-independent PKC activity; in addition, increases in both initial and total PKA activities were also recorded. The effect of okadaic acid on noradrenaline-stimulated 3H-IP accumulation were not, however, mimicked by either forskolin or 8-bromo-cyclic AMP, suggesting that this event is not regulated by PKA. Our data point to roles for both PKC and PP2A in the regulation of alpha(1A)-adrenoceptor-linked phosphoinositide metabolism in cultured cortical glia.     

Myosin II and Rho kinase activity are required for melanosome aggregation in fish retinal pigment epithelial cells

In the retinal pigment epithelium (RPE) of fish, melanosomes (pigment granules) migrate long distances through the cell body into apical projections in the light, and aggregate back into the cell body in the dark. RPE cells can be isolated from the eye, dissociated, and cultured as single cells in vitro. Treatment of isolated RPE cells with cAMP or the phosphatase inhibitor, okadaic acid (OA), stimulates melanosome aggregation, while cAMP or OA washout in the presence of dopamine triggers dispersion. Previous studies have shown that actin filaments are both necessary and sufficient for aggregation and dispersion of melanosomes within apical projections of isolated RPE. The role of myosin II in melanosome motility was investigated using the myosin II inhibitor, blebbistatin, and a specific rho kinase (ROCK) inhibitor, H-1152. Blebbistatin and H-1152 partially blocked melanosome aggregation triggered by cAMP in dissociated, isolated RPE cells and isolated sheets of RPE. In contrast, neither drug affected melanosome dispersion. In cells exposed to either blebbistatin or H-1152, then triggered to aggregate using OA, melanosome aggregation was completely inhibited. These results demonstrate that (1) melanosome aggregation and dispersion occur through different, actin-dependent mechanisms; (2) myosin II and ROCK activity are required for full melanosome aggregation, but not dispersion; (3) partial aggregation that occurred despite myosin II or ROCK inhibition suggests a second component of aggregation that is dependent on cAMP signaling, but independent of ROCK and myosin II.     

Regulation of bidirectional melanosome transport by organelle bound MAP kinase

Regulation of intracellular transport plays a role in a number of processes, including mitosis, determination of cell polarity, and neuronal growth. In Xenopus melanophores, transport of melanosomes toward the cell center is triggered by melatonin, whereas their dispersion throughout the cytoplasm is triggered by melanocyte-stimulating hormone (MSH), with both of these processes mediated by cAMP-dependent protein kinase A (PKA) activity [1, 2]. Recently, the ERK (extracellular signal-regulated kinase) pathway has been implicated in regulating organelle transport and signaling downstream of melatonin receptor [3, 4]. Here, we directly demonstrate that melanosome transport is regulated by ERK signaling. Inhibition of ERK signaling by the MEK (MAPK/ERK kinase) inhibitor U0126 blocks bidirectional melanosome transport along microtubules, and stimulation of ERK by constitutively active MEK1/2 stimulates transport. These effects are specific because perturbation of ERK signaling has no effect on the movement of lysosomes, organelles related to melanosomes [5]. Biochemical analysis demonstrates that MEK and ERK are present on melanosomes and transiently activated by melatonin. Furthermore, this activation correlates with an increase in melanosome transport. Finally, direct inhibition of PKA transiently activates ERK, demonstrating that ERK acts downstream of PKA. We propose that signaling of organelle bound ERK is a key pathway that regulates bidirectional, microtubule-based melanosome transport.     

Smoothened activates Galphai-mediated signaling in frog melanophores

The 7-pass transmembrane protein Smoothened was investigated for its ability to act as a G-protein-coupled receptor in Xenopus laevis melanophores. A plasmid containing the human Smoothened cDNA insert was transfected into immortalized frog pigment cells. Cells expressing the protein showed a phenotype of persistent pigment aggregation, a hallmark of constitutive Galpha(i) activation. Smoothened-mediated pigment aggregation was reversed by treatment with pertussis toxin or by co-expression with dominant negative Galpha(i). The ability of melanophores to express functional Smoothened was also determined by its co-expression with the twelve-pass transmembrane protein, Patched. Patched blocked Smoothened-mediated melanosome aggregation in a dose-dependent manner, consistent with its physiological role as an inhibitor of Smoothened. That the reconstituted Patched-Smoothened receptor complex functions normally in pigment cells was demonstrated by co-transfection with the activating ligand, Sonic hedgehog, as well as by direct application of the recombinant Sonic hedgehog protein. Sonic hedgehog reversed Patched-mediated inhibition of Smoothened and induced pigment aggregation. The findings demonstrate that the human Sonic hedgehog receptor complex can be functionally reconstituted in melanophores and that it is capable of transmembrane signaling by utilizing endogenous Galpha(i).     

Heterotrimeric Kinesin II Is the Microtubule Motor Protein Responsible for Pigment Dispersion in Xenopus Melanophores

Melanophores move pigment organelles (melanosomes) from the cell center to the periphery and vice-versa. These bidirectional movements require cytoplasmic microtubules and microfilaments and depend on the function of microtubule motors and a myosin. Earlier we found that melanosomes purified from Xenopus melanophores contain the plus end microtubule motor kinesin II, indicating that it may be involved in dispersion (Rogers, S.L., I.S. Tint, P.C. Fanapour, and V.I. Gelfand. 1997. Proc. Natl. Acad. Sci. USA. 94: 3720–3725). Here, we generated a dominant-negative construct encoding green fluorescent protein fused to the stalk-tail region of Xenopus kinesin-like protein 3 (Xklp3), the 95-kD motor subunit of Xenopus kinesin II, and introduced it into melanophores. Overexpression of the fusion protein inhibited pigment dispersion but had no effect on aggregation. To control for the specificity of this effect, we studied the kinesin-dependent movement of lysosomes. Neither dispersion of lysosomes in acidic conditions nor their clustering under alkaline conditions was affected by the mutant Xklp3. Furthermore, microinjection of melanophores with SUK4, a function-blocking kinesin antibody, inhibited dispersion of lysosomes but had no effect on melanosome transport. We conclude that melanosome dispersion is powered by kinesin II and not by conventional kinesin. This paper demonstrates that kinesin II moves membrane-bound organelles.     

Relationship Between the Structure of Sangivamycin-Derived Nucleosides and Their Effect on Leukemic Cell Growth and on Protein Kinase A and C Activity

Abstract

The nucleoside antibiotic sangivamycin (4-amino-7-(β-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine-5-carboxamide, (1) is an effective inhibitor of protein kinase A (PKA) and protein kinase C (PKC) but, upon its phosphorylation in intact cells, it gains the ability to affect other targets as well. To retain its selectivity for the protein kinases, a series of nonphosphorylatable sangivamycin derivatives was prepared by replacing the 5′-hydroxyl group with other functions including N₃, F, SO₂NH₂, NO₂, and NH₂, These derivatives were more potent inhibitors of PKA and PKC than were the phosphorylatable compounds, although the latter were more potent inhibitors of leukemic cell growth.     

佛波酯引起蛋白激酶C下降调节的专一性

探讨了佛波酯（ＰＭＡ）对蛋白激酶的下降调节是否有激酶专一性及亚型专一性．用组蛋白Ｈ１作为蛋白激酶Ｃ（ＰＫＣ）和蛋白激酶Ａ（ＰＫＡ）的受体底物,加入ＰＫＣ和ＰＫＡ的特异性激活剂区分ＰＫＣ和ＰＫＡ,用聚谷酪（４１）为酪氨酸蛋白激酶（ＴＰＫ）的专一性受体底物,以３２Ｐ－ＡＴＰ为３２Ｐ共同供体底物测定三种蛋白激酶的活力,并用免疫组化法测定ＰＫＣ亚型．结果发现ＰＭＡ对人７７２１肝癌细胞只引起ＰＫＣ而不引起ＰＫＡ和ＴＰＫ的下降调节,ＰＫＣ的非特异性抑制剂槲皮素和特异性抑制剂Ｄ－鞘氨醇能大部分取消ＰＭＡ对ＰＫＣ的下降调节,但ＴＰＫ抑制剂ｇｅｎｅｓｔｅｉｎ则没有阻断下降调节的作用．用ＨＬ－６０细胞还证明ＰＭＡ只对含量丰富的ＰＫＣα和ＰＫＣβⅡ亚型而不对含量很少的ＰＫＣβⅠ亚型发生下降调节．上述结果说明ＰＭＡ对蛋白激酶的下降调节有激酶和亚型专一性．     

Protein phosphatase 2A regulates deoxycytidine kinase activity via Ser-74 dephosphorylation

Deoxycytidine kinase (dCK) is a critical enzyme for activation of anticancer nucleoside analogs. Its activity is controlled via Ser-74 phosphorylation. Here, we investigated which Ser/Thr phosphatase dephosphorylates Ser-74. In cells, the PP1/PP2A inhibitor okadaic acid increased both dCK activity and Ser-74 phosphorylation at concentrations reported to specifically target PP2A. In line with this, purified PP2A, but not PP1, dephosphorylated recombinant pSer-74-dCK. In cell lysates, the Ser-74-dCK phosphatase activity was found to be latent, Mn²⁺-activated, responsive to PP2A inhibitors, and diminished after PP2A-immunodepletion. Use of siRNAs allowed concluding definitively that PP2A constitutively dephosphorylates dCK in cells and negatively regulates its activity.