Casein kinase 1δ activates human recombinant deoxycytidine kinase by Ser-74 phosphorylation, but is not involved in the in vivo regulation of its activity

Deoxycytidine kinase (dCK) is a key enzyme in the salvage of deoxynucleosides and in the activation of several anticancer and antiviral nucleoside analogues. We recently showed that dCK was activated in vivo by phosphorylation of Ser-74. However, the protein kinase responsible was not identified. Ser-74 is located downstream a Glu-rich region, presenting similarity with the consensus phosphorylation motif of casein kinase 1 (CKI), and particularly of CKI δ. We showed that recombinant CKI δ phosphorylated several residues of bacterially overexpressed dCK: Ser-74, but also Ser-11, Ser-15, and Thr-72. Phosphorylation of dCK by CKI δ correlated with increased activity reaching at least 4-fold. Site-directed mutagenesis demonstrated that only Ser-74 phosphorylation was involved in dCK activation by CKI δ, strengthening the key role of this residue in the control of dCK activity. However, neither CKI δ inhibitors nor CKI δ siRNA-mediated knock-down modified Ser-74 phosphorylation or dCK activity in cultured cells. Moreover, these approaches did not prevent dCK activation induced by treatments enhancing Ser-74 phosphorylation. Taken together, the data preclude a role of CKI δ in the regulation of dCK activity in vivo. Nevertheless, phosphorylation of dCK by CKI δ could be a useful tool for elucidating the influence of Ser-74 phosphorylation on the structure-activity relationships in the enzyme.     

Phosphorylation of theα subunit of the sodium channel by protein kinase C

The alpha subunit of the purified voltage-sensitive sodium channel from rat brain is rapidly phosphorylated to the extent of 3-4 mol phosphate/mol by purified protein kinase C. The alpha subunit of the native sodium channel in synaptosomal membranes is also phosphorylated by added protein kinase C as assessed by specific immunoprecipitation and polyacrylamide gel electrophoresis of labeled membranes. Our results suggest coordinate regulation of sodium channel phosphorylation state by cAMP-dependent and calcium/phospholipid-dependent protein kinases.     

Functional role of the charge at the T538 residue in the control of protein kinase Cθ

We show that protein kinase C (PKC) θ localized at the Golgi complex is partially conjugated to monoubiquitin. Using the inactive T538A and activable T538E mutants of PKCθ, we demonstrate that the presence of an uncharged residue at the 538 position of the activation loop favors both association with the Golgi and monoubiquitination of the kinase. Moreover, the inactive PKCθ does not translocate from the Golgi in response to a short-term cell stimulation with a phorbol ester and is subjected to different proteolytic degradation pathways compared to the activable cytosolic kinase. These findings highlight the role of T538 as a critical determinant to address the activable and the inactive PKCθ molecules to different intracellular compartments and to specific post-transductional modifications. The functional relevance of these observations is supported by the impaired cell division observed in phenotypes expressing high levels of the inactive PKCθ.     

Functional significance of the shark Na,K-ATPase N-terminal domain. Is the structurally variable N-Terminus involved in tissue-specific regulation by FXYD proteins?

The proteolytic profile after mild controlled trypsin cleavage of shark rectal gland Na,K-ATPase was characterized and compared to that of pig kidney Na,K-ATPase, and conditions for achieving N-terminal cleavage of the alpha-subunit at the T(2) trypsin cleavage site were established. Using such conditions, the shark enzyme N-terminus was much more susceptible to proteolysis than the pig enzyme. Nevertheless, the maximum hydrolytic activity was almost unaffected for the shark enzyme, whereas it was significantly decreased for the pig kidney enzyme. The apparent ATP affinity was unchanged for shark but increased for pig enzyme after N-terminal truncation. The main common effect following N-terminal truncation of shark and pig Na,K-ATPase is a shift in the E(1)-E(2) conformational equilibrium toward E(1). The phosphorylation and the main rate-limiting E(2) --> E(1) step are both accelerated after N-terminal truncation of the shark enzyme, but decreased significantly in the pig kidney enzyme. Some of the kinetic differences, like the acceleration of the phosphorylation reaction, following N-terminal truncation of the two preparations may be due to the fact that under the conditions used for N-terminal truncation, the C-terminal domain of the FXYD regulatory protein of the shark enzyme, PLMS or FXYD10, was also cleaved, whereas the gamma or FXYD2 of the pig enzyme was not. In the shark enzyme, N-terminal truncation of the alpha-subunit abolished association of exogenous PLMS with the alpha-subunit and the functional interactions were abrogated. Moreover, PKC phosphorylation of the preparation, which relieves PLMS inhibition of Na,K-ATPase activity, exposed the N-terminal trypsin cleavage site. It is suggested that PLMS interacts functionally with the N-terminus of the shark Na,K-ATPase to control the E(1)-E(2) conformational transition of the enzyme and that such interactions may be controlled by regulatory protein kinase phosphorylation of the N-terminus. Such interactions are likely in shark enzyme where PLMS has been demonstrated by cross-linking to associate with the Na,K-ATPase A-domain.     

Immunological detection of the mitochondrial F1-ATPase alpha subunit in the matrix of rat liver peroxisomes. A protein involved in organelle biogenesis?

Rat liver peroxisomes contain in their matrix the alpha-subunit of the mitochondrial F1-ATPase complex. The identification of this protein in liver peroxisomes has been achieved by immunoelectron microscopy and subcellular fractionation. No beta-subunit of the mitochondrial F1-ATPase complex was detected in the peroxisomal fractions obtained in sucrose gradients or in Nycodenz pelletted peroxisomes. The consensus peroxisomal targeting sequence (Ala-Lys-Leu) is found at the carboxy terminus of the mature alpha-subunit from bovine heart and rat liver mitochondria. Due to the dual subcellular localization of the alpha-subunit and to the structural homologies that exist between this protein and molecular chaperones [(1990) Biol. Chem. 265, 7713-7716] it is suggested that the protein should perform another functional role(s) in both organelles, plus to its characteristic involvement in the regulation of mitochondrial ATPase activity.     

α-tocopherol modulates tyrosine phosphorylation in human neutrophils by inhibition of protein kinase C activity and activation of tyrosine phosphatases

α-Tocopherol augmentation in human neutrophils was investigated for effects on neutrophil activation and tyrosine phosphorylation of proteins, through its modulation of protein kinase C (PKC) and tyrosine phosphatase activities. Incubation of neutrophils with α-tocopherol succinate (TS) resulted in a dose-dependent incorporation into cell membranes, up to 2.5 nmol/2 × 10⁶ cells. A saturating dose of TS (40 μmol/l) inhibited oxidant production by neutrophils stimulated with phorbol myristate acetate (PMA) or opsonized zymosan (OZ) by 86 and 57%, as measured by luminol-amplified chemiluminescence (CL). With PMA, TS inhibited CL generation to a similar extent to staurosporine (10 nmol/l) or genistein (100 μmol/l), and much more than Trolox (40 μmol/l). With OZ, TS inhibited CL to a similar extent to Trolox. Neutrophil PKC activity was inhibited 50% or more by TS or staurosporine. The enzyme activity was unaffected by genistein or Trolox, indicating a specific interaction of α-tocopherol. TS or Trolox increased protein tyrosine phosphorylation in resting neutrophils, and as with staurosporine further increased tyrosine phosphorylation in PMA-stimulated neutrophils, while the tyrosine kinase (TK) inhibitor genistein diminished phosphorylation. These effects in resting or PMA-stimulated neutrophils were unrelated to protein tyrosine phosphatase (PTP) activities, which were maintained or increased by TS or Trolox. In OZ-stimulated neutrophils, on the other hand, all four compounds inhibited the increase in tyrosine-phosphorylated proteins. In this case, the effects of pre-incubation with TS or Trolox corresponded with partial inhibition of the marked (85%) decrease in PTP activity induced by OZ. These results indicate that α-tocopherol inhibits PMA-activation of human neutrophils by inhibition of PKC activity, and inhibits tyrosine phosphorylation and activation of OZ-stimulated neutrophils also through inhibition of phosphatase inactivation.     

Is cholinergic activity of the caudate nucleus involved in memory?

A review was made of experiments dealing with the involvement of cholinergic activity of the caudate nucleus in memory processes. Injections of acetylcholine-receptor blockers or of neurotoxins against cholinergic interneurons into the striatum produce marked impairments in acquisition and retention of instrumental tasks while injections of acetylcholine or choline into the caudate produce the opposite effect. However, after a period of overtraining cholinergic blockade or interference with neural activity of the caudate does not produce significant deficits in retention. It is concluded that striatal cholinergic activity is critically involved in memory of recent events and that long-term memory is mediated by different neurochemical systems outside the caudate nucleus.     

Evidence for the phosphorylation of serine259 of histone deacetylase 5 by protein kinase Cδ

Signaling via pro-growth G protein coupled receptors triggers phosphorylation of HDAC5 on two serine residues (Ser₂₅₉ and Ser₄₉₈), resulting in nuclear export of HDAC5 and de-repression of downstream target genes. In the previous paper we reported the important role of PKD isozymes in the regulation of HDAC5 by phosphorylating Ser₄₉₈ of HDAC5 [Q.K. Huynh, T.A. Mckinsey, Arch. Biochem. Biophys. 450 (2006) 141–148]. In the present paper, we provide evidence that PKCδ can directly phosphorylate Ser₂₅₉ of HDAC5. The evidence is based on the following facts (a) isolated kinase fraction from human failing heart tissues contained PKCδ that phosphorylated HDAC5 Ser₂₅₉ peptide and no significant activity was found for the unbound fraction after they were immunoprecipitated with PKCδ specific antibody; (b) specific inhibitors for PKCδ inhibited kinase activity from isolated fraction and recombinant human PKCδ with similar IC₅₀ values; (c) recombinant human PKCδ can directly phosphorylate full length Ser₂₅₉ HDAC5 protein and HDAC5 Ser₂₅₉ peptide. The results suggest that in addition to activation of protein kinase D isozymes by phosphorylating Ser₇₄₄ and Ser₇₄₈ at their activation sites, PKCδ may also play a role in the regulation of HDAC5 by phosphorylation of Ser₂₅₉.     

What is the correct value for Na,K-ATPase activity in homogenates of cerebral cortex?

Activity of Na, K-ATPase in homogenates of fresh cerebral cortex of rats was compared with that of cortex frozen under different conditions. Activity yields after rapid in situ freezing of the exposed cerebral cortex were twice, higher (26.1 U) than in homogenates of the fresh cortex (13.3 U). Fresh brain kept on ice for 60 and 300 s and subsequently frozen in liquid nitrogen yielded activities comparable to those of the tissue frozen in situ (24.1 U and 24.9 U for 60 s and 300 s periods, respectively). Inhibition of Na, K-ATPase by 10⁻⁷ M vanadate was significantly stronger (38%) in homogenates of the fresh brain then in those of the cortex frozen in situ (28%). High Na, K-ATPase activity (47.6 U) in suspensions of synaptosomal membranes (SM) prepared from fresh cortical homogenates was only slightly inhibited by 10⁻⁷ M vanadate (12%). Various treatments of homogenates or SM suspensions, like increase of piston rotation speed, repeated freezing and thawing procedure or vigorous shaking did not significantly affect the enzyme activity. Mg-ATPase activity and its sensitivity to vanadate was also modified by tissue treatment but the effect was much less pronounced.     

Cyclin G-associated kinase regulates protein phosphatase 2A by phosphorylation of its B'γ subunit

Protein phosphatase 2A (PP2A) bearing the B’γ (= B’α/B56γ1/PR61γ) subunit is recruited to dephosphorylation targets by cyclin G. We demonstrate here that cyclin G-associated kinase (GAK), a component of the GAK/B’γ/cyclin G complex, directly phosphorylates the B’γ-Thr104 residue and regulates PP2A activity. Indeed, an anti-B’γ-pT104 antibody detected immunofluorescence signals at the chromosome and centrosome during mitosis; these signals were reduced by siRNA-mediated GAK knockdown. After DNA damage by γ-irradiation, the chromosome signals formed foci that colocalized with a DNA double-strand break (DSB) marker H2AX-pS139 (γH2AX) and CHK2-pT68. Moreover, B’γ-pT104 enhanced PP2A holoenzyme assembly and PP2A activity, as shown by the results of an in vitro phosphatase assay. These results suggest a novel role for GAK as a regulator of dephosphorylation events under the control of the PP2A B’γ subunit.     

Cytochemical and immunocytochemical localization of Na,K-ATPase α subunit isoenzymes in the rat heart

In order to understand the functional significance of Na,K-ATPase subunits as well as their isoenzymes, a precise subcellular localization of these in the myocyte is a crucial prerequisite. Cytochemical, immunofluorescence, preembedding immunogold and horse radish peroxidasediaminobenzidine methods, demonstrated ₁ isoenzyme immunoreactivity on the sarcolemma, T-tubules and the subsarcolemmal cisterns of the adult cardiac myocytes. Cytochemically, ouabain resistant Na,K-ATPase precipitate was localized only in the subsarcolemmal cisterns and junctional sarcoplasmic reticulum. For ₂ isoenzyme, immunoreactivity was demonstrated on the sarcolemma as well as in all areas of the myocytes in particularly a close proximation to the sarcoplasmic reticulum and microsomes. For ₃ isoenzyme, only a weak insignificant signal was noted on the sarcolemma, intercalated disc and sarcoplasm. It is suggested that cytochemical ouabain resistant precipitate present in subsarcolemmal cisterns and junctional sarcoplasmic reticulum represent ₁ isoenzyme of Na,K-ATPase. A differential as well as unique localization of subunit isoenzymes of Na,K-ATPase in specific structures of cardiac myocytes may suggest importance in physiological function at these sites.     

Is the subunit the minimal function unit of creatine kinase?

The dimeric rabbit muscle isozyme of creatine kinase (MM) is modified by iodoacetamide to produce the inactive dimer (M'M') and then hybridized with native dimeric brain isozyme (BB). The hybrid enzyme (M'B), as isolated by PAGE, has the same Km for both ATP and creatine but half the specific activity of the brain isozyme (BB). Likewise, the hybrid of the modified brain with the native muscle isozyme (MB') has half the activity of the native muscle enzyme. The M'B, MB' and MB hybrid dimers all have essentially the same electrophoretic properties, and their intrinsic fluorescence and CD spectra in the far-ultraviolet region are very similar to those of the homodimers MM and BB. Similar results were obtained for the hybrid (M"B) containing the muscle enzyme subunit modified at both the thiol group with iodoacetamide and the Trp residue with dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide and the native brain enzyme submit. The above results suggest strongly the independent catalytic function of the subunit of creatine kinase.     

What is the correct value for Na, K-ATPase activity in homogenates of cerebral cortex?

Activity of Na, K-ATPase in homogenates of fresh cerebral cortex of rats was compared with that of cortex frozen under different conditions. Activity yields after rapid in situ freezing of the exposed cerebral cortex were twice, higher (26.1 U) than in homogenates of the fresh cortex (13.3 U). Fresh brain kept on ice for 60 and 300 s and subsequently frozen in liquid nitrogen yielded activities comparable to those of the tissue frozen in situ (24.1 U and 24.9 U for 60 s and 300 s periods, respectively). Inhibition of Na, K-ATPase by 10(-7) M vanadate was significantly stronger (38%) in homogenates of the fresh brain then in those of the cortex frozen in situ (28%). High Na, K-ATPase activity (47.6 U) in suspensions of synaptosomal membranes (SM) prepared from fresh cortical homogenates was only slightly inhibited by 10(-7) M vanadate (12%). Various treatments of homogenates or SM suspensions, like increase of piston rotation speed, repeated freezing and thawing procedure or vigorous shaking did not significantly affect the enzyme activity. Mg-ATPase activity and its sensitivity to vanadate was also modified by tissue treatment but the effect was much less pronounced.     

Diacylglycerol kinase α enhances protein kinase Cζ-dependent phosphorylation at Ser311 of p65/RelA subunit of nuclear factor-κB

We recently reported that diacylglycerol kinase (DGK) α enhanced tumor necrosis factor-α (TNF-α)-induced activation of nuclear factor-κB (NF-κB). However, the signaling pathway between DGKα and NF-κB remains unclear. Here, we found that small interfering RNA-mediated knockdown of DGKα strongly attenuated protein kinase C (PKC) ζ-dependent phosphorylation of a large subunit of NF-κB, p65/RelA, at Ser311 but not PKCζ-independent phosphorylation at Ser468 or Ser536. Moreover, knockdown and overexpression of PKCζ suppressed and synergistically enhanced DGKα-mediated NF-κB activation, respectively. These results strongly suggest that DGKα positively regulates TNF-α-dependent NF-κB activation via the PKCζ-mediated Ser311 phosphorylation of p65/RelA.     

Nox1 activation by βPix and the role of Ser-340 phosphorylation

Rac is an activating factor for Nox1, an O₂⁻-generating NADPH oxidase, expressed in the colon and other tissues. Rac requires a GDP-GTP exchange factor for activation. Nox1 activation by βPix has been demonstrated in cell lines. We examined the effects of βPix and its phosphomimetic mutant on endogenous Nox1 in Caco-2 cells transfected with Noxo1 and Noxa1. βPix expression enhanced O₂⁻ production in resting cells and cells stimulated with EGF or phorbol ester. βPix(S340E) further enhanced O₂⁻ production, while βPix(S340A) eliminated the βPix effect. βPix(S340E), but not βPix(S340A), had higher affinity and GEF activity for Rac than wild-type βPix. These results suggest that βPix phosphorylation at Ser-340 upregulates Nox1 through Rac activation, confirming Rac as a trigger for acute Nox1-dependent ROS production.     

Is there a link between impaired glucose metabolism and protein kinase C activity in the diabetic heart?

The activity of the isoform of protein kinase C (PKC) is reduced in the diabetic heart. Since this isozyme has been implicated in insulin action, we tested the hypothesis that PKC contributes to the development of impaired glucose metabolism by the noninsulin-dependent diabetic heart. Exposure of the diabetic heart to buffer containing the protein kinase C activator, phorbol myristate acetate, increased PKC activity in the membrane. Associated with the improvement in PKC activity was a biphasic change in glucose metabolism. The initial phase was characterized by a breakdown in glycogen stores, a stimulation in glucose oxidation and a decrease in endogenous fatty acid oxidation. This was followed by a second phase in which the uptake of glucose was modestly stimulated. Nonetheless, since the phorbol ester did not overcome the diabetes-linked defect in pyruvate dehydrogenase, the increase in glycolytic flux was not associated with a rise in glucose oxidation. Consequently, nearly 50% of the triose units were diverted into lactate and pyruvate production and the generation of ATP from glucose was restricted. Since insulin promotes not only glucose uptake, but also glycogen synthesis and glucose oxidation, the phorbol ester and insulin effects are very different. Thus, the data do not support a role for PKC in the development of glucose metabolic defects in the hearts of noninsulin-dependent diabetic rats.