Endogenous Protein Kinase Research Articles

Dependence of aminoglycoside 3′-phosphotransferase VIII activity on serine/threonine protein kinases in Streptomyces rimosus

In Streptomyces rimosus, selection for resistance to the aminoglycoside antibiotic kanamycin triggers the normally silent aminoglycoside 3′-phosphotransferase VIII gene (aphVIII). The expression of APHVIII is accompanied by amplification of the chromosomal DNA fragment containing the aphVIII gene. Earlier, S. rimosus aphVIII gene was isolated and sequenced. Using in vitro labeling and immunoprecipitation with anti-APHVIII antibodies, we have demonstrated that endogenous protein kinases (PKs) in extracts of S. rimosus strain S683 actively phosphorylate two serine residues in the APHVIII molecule. The amount of phosphate incorporated into APHVIII in the presence of Ca2+ is 1.84-fold greater than that without Ca2+. Analysis of ingel autophosphorylation and phosphorylation of the substrate incorporated into the gel matrix has shown that modification of APHVIII is catalyzed by two serine/threonine PKs (74 kDa and 55 kDa). The activity of 55-kDa PK is dependent on Ca2+ and calmodulin. The specific kanamycin phosphotransferase activity of exhaustively phosphorylated APHVIII is 3.72 times higher than that of the unmodified enzyme. It is proposed that the above PKs may be involved in the regulation of kanamycin resistance in S. rimosus.

Purified Bacillus anthracis Lethal Toxin Complex Formed in Vitro and during Infection Exhibits Functional and Biological Activity

Anthrax protective antigen (PA, 83 kDa), a pore-forming protein, upon protease activation to 63 kDa (PA(63)), translocates lethal factor (LF) and edema factor (EF) from endosomes into the cytosol of the cell. The relatively small size of the heptameric PA(63) pore (approximately 12 angstroms) raises questions as to how large molecules such as LF and EF can move through the pore. In addition, the reported high binding affinity between PA and EF/LF suggests that EF/LF may not dissociate but remain complexed with activated PA(63). In this study, we found that purified (PA(63))(7)-LF complex exhibited biological and functional activities similar to the free LF. Purified LF complexed with PA(63) heptamer was able to cleave both a synthetic peptide substrate and endogenous mitogen-activated protein kinase kinase substrates and kill susceptible macrophage cells. Electrophysiological studies of the complex showed strong rectification of the ionic current at positive voltages, an effect similar to that observed if LF is added to the channels formed by heptameric PA(63) pore. Complexes of (PA(63))(7)-LF found in the plasma of infected animals showed functional activity. Identifying active complex in the blood of infected animals has important implications for therapeutic design, especially those directed against PA and LF. Our studies suggest that the individual toxin components and the complex must be considered as critical targets for anthrax therapeutics.

Activation of SIPK in response to UV-C irradiation: utility of a glutathione-S transferase-tagged plant MAP kinase by transient expression with agroinfiltration

Salicylic acid (SA)-induced protein kinase (SIPK) in Nicotiana tabacum L. is activated in response to various stimuli. We assessed the response of SIPK in N. benthamiana. leaves to ultraviolet-C (UV-C) and SA stress. Infiltration of Agrobacterium tumefaciens EHA105 cells containing pBE2113-GST-SIPK into leaves gave transient production of glutathione-S transferase (GST)-fused SIPK. Irradiation of agroinfiltrated leaves with UV-C (254 nm) activated GST-SIPK and endogenous protein kinases with a molecular mass of about 45 kDa, close to that of SIPK simultaneously. These results suggest that SIPK is involved in stress signalling in Nicotiana plants under UV-C irradiation.

Protein Kinase A Activity And Protein Phosphorylation During The Mouse Sperm Acrosomal Reaction

Using two-dimensional gel electrophoresis, changes in protein phosphorylation caused by cyclic nucleotide-dependent protein kinases were analyzed with or without exposure to a protein kinase inhibitor, H-8, during the mouse sperm acrosomal reaction. The acrosomal reaction, induced by the treatment of sperm with dibutyryl cyclic AMP or dibutyryl cyclic GMP, was inhibited by H-8. The activities of cyclic AMP-dependent protein kinase (PKA) and cyclic GMP-dependent protein kinase induced by the sperm extract were also inhibited by H-8. When endogenous PKA in sperm was activated by the addition of cyclic AMP, a 45-kDa protein spot identified by electrophoresis indicated the occurrence of phosphorylation in vivo. Furthermore, the enhanced phosphorylation of the 45-kDa protein spot was inhibited by H-8. These results suggest that the PKA-catalyzed phosphorylation of the 45-kDa protein may be involved in the regulation of the mouse sperm acrosomal reaction.

Endogenous Protein Kinase Inhibitor γ Terminates Immediate-early Gene Expression Induced by cAMP-dependent Protein Kinase (PKA) Signaling: TERMINATION DEPENDS ON PKA INACTIVATION RATHER THAN PKA EXPORT FROM THE NUCLEUS

Expression of many genes induced by cAMP-dependent protein kinase (PKA) signaling is rapidly terminated. Although many mechanisms contribute to regulation of PKA signaling, members of the endogenous protein kinase inhibitor (PKI) family may be particularly important for terminating nuclear PKA activity and gene expression. Here we used both siRNA and antisense knockdown strategies to examine PKA signaling activated by parathyroid hormone or the beta-adrenergic agonist, isoproterenol. We found that endogenous PKIgamma is required for efficient termination of nuclear PKA activity, transcription factor phosphorylation, and immediate-early genes. Moreover, PKIgamma is required for export of PKA catalytic subunits from the nucleus back to the cytoplasm following activation of PKA signaling because this is also inhibited by PKIgamma knockdown. Leptomycin B blocks PKA nuclear export but has little or no effect on nuclear PKA activity or immediate-early gene expression. Thus, inactivation of PKA activity in the nucleus is sufficient to terminate signaling, and nuclear export is not required. These results were the first in any cell type showing that endogenous levels of PKI regulate PKA signaling.

Enhanced Dephosphorylation of cAMP-dependent Protein Kinase by Oxidation and Thiol Modification

The catalytic subunit of cAMP-dependent protein kinase (PKA) is phosphorylated at threonine 197 and serine 338. Phosphorylation of threonine 197, located in the activation loop, is required for coordinating the active site conformation and optimal enzymatic activity. However, this phosphorylation has not been widely appreciated as a regulatory site because of the apparent constitutive nature of the phosphorylation and the general resistance of the kinase to phosphatase treatment. We demonstrate here that the observed resistance of the catalytic subunit to dephosphorylation is due, in part, to the presence of the highly nucleophilic cysteine 199 located proximal to the phosphate on threonine 197. Experiments performed in vitro demonstrated that mutation (cysteine 199 to alanine), oxidation, such as by glutathionylation or internal disulfide bond formation, or alkylation of the C-subunit enhanced its ability to be dephosphorylated. Furthermore, rephosphorylation of reduced C-subunit by PDK1 created a cycle whereby the inactive kinase could be reactivated. To demonstrate that thiol modification of PKA can lead to enhanced dephosphorylation in vivo, PC12 cells were treated with N-ethylmaleimide (NEM). Such treatment resulted in complete PKA inactivation and dephosphorylation of threonine 197. This effect of NEM was contingent upon prior treatment of the cells with PKA activators, demonstrating the resistance of the holoenzyme to thiol alkylation-mediated dephosphorylation. Our results also demonstrated that NEM treatment of PC12 cells enhanced the dephosphorylation of the protein kinase Calpha activation loop, suggesting a common mechanism of regulation among members of the AGC family of kinases.

PKCδ and MAPK mediate G 1 arrest induced by PMA in SKBR-3 breast cancer cells

The effects of activating endogenous protein kinase C (PKC) on cell proliferation and the cell cycle were investigated by treating the breast cancer cell line SKBR-3 with phorbol 12-myristate 13 acetate (PMA). This inhibited cell growth in a concentration-dependent manner, causing a marked arrest of cells in G 1. Pre-treatment with GF109203X completely blocked the antiproliferative effect of PMA, and pre-treatment with the PKCδ inhibitor rottlerin partially blocked it. Infecting SKBR-3 cells with an adenovirus vector containing wild-type PKCδ, WTPKCδAdV, had similar effects on PMA. Infecting the cells with a dominant-negative PKCδAdV construct blocked the growth inhibition induced by PMA. Downstream of PKC, PMA treatment inhibited extracellular signal-regulated kinase mitogen-activated protein kinase phosphorylation, up-regulated c-jun NH 2-terminal kinase phosphorylation, and inhibited retinoblastoma (Rb) phosphorylation. These results strongly implicated PKC (mainly PKCδ) in the G 1 arrest induced by PMA and suggested PKC as a target for breast cancer treatment.

Alterations in Myofilament Function Contribute to Left Ventricular Dysfunction in Pigs Early After Myocardial Infarction

Myocardial infarction (MI) initiates cardiac remodeling, depresses pump function, and predisposes to heart failure. This study was designed to identify early alterations in Ca2+ handling and myofilament proteins, which may contribute to contractile dysfunction and reduced beta-adrenergic responsiveness in postinfarct remodeled myocardium. Protein composition and contractile function of skinned cardiomyocytes were studied in remote, noninfarcted left ventricular (LV) subendocardium from pigs 3 weeks after MI caused by permanent left circumflex artery (LCx) ligation and in sham-operated pigs. LCx ligation induced a 19% increase in LV weight, a 69% increase in LV end-diastolic area, and a decrease in ejection fraction from 54+/-5% to 35+/-4% (all P<0.05), whereas cardiac responsiveness to exercise-induced increases in circulating noradrenaline levels was blunted. Endogenous protein kinase A (PKA) was significantly reduced in remote myocardium of MI animals, and a negative correlation (R=0.62; P<0.05) was found between cAMP levels and LV weight-to-body weight ratio. Furthermore, SERCA2a expression was 23% lower after MI compared with sham. Maximal isometric force generated by isolated skinned myocytes was significantly lower after MI than in sham (15.4+/-1.5 versus 19.2+/-0.9 kN/m2; P<0.05), which might be attributable to a small degree of troponin I (TnI) degradation observed in remodeled postinfarct myocardium. An increase in Ca2+ sensitivity of force (pCa50) was observed after MI compared with sham (DeltapCa50=0.17), which was abolished by incubating myocytes with exogenous PKA, indicating that the increased Ca2+ sensitivity resulted from reduced TnI phosphorylation. In conclusion, remodeling of noninfarcted pig myocardium is associated with decreased SERCA2a and myofilament function, which may contribute to depressed LV function. The full text of this article is available online at http://circres.ahajournals.org.

Rac1 inhibits apoptosis in human lymphoma cells by stimulating Bad phosphorylation on Ser-75.

The small GTPase Rac1 has emerged as an important regulator of cell survival and apoptosis, but the mechanisms involved are not completely understood. In this report, constitutively active Rac1 is shown to stimulate the phosphorylation of the Bcl-2 family member Bad, thereby suppressing drug-induced caspase activation and apoptosis in human lymphoma cells. Rac1 activation leads to human Bad phosphorylation specifically at serine-75 (corresponding to murine serine-112) both in vivo and in vitro. Inhibition of constitutive and activated Rac1-induced Bad phosphorylation by a cell-permeable competitive peptide inhibitor representing this Bad phosphorylation site sensitizes lymphoma cells to drug-induced apoptosis. The data show further that endogenous protein kinase A is a primary catalyst of cellular Bad phosphorylation in response to Rac activation, while Akt is not involved. These findings define a mechanism by which active Rac1 promotes lymphoma cell survival and inhibits apoptosis in response to cancer chemotherapy drugs.

Bromodomain factor 1 (Bdf1) is phosphorylated by protein kinase CK2.

Bromodomain factor 1 (Bdf1) associates with Saccharomyces cerevisiae TFIID and corresponds to the C-terminal half of higher eukaryotic TAF1. It also associates with the SWR-C complex, which is important for Htz1 deposition. Bdf1 binds preferentially to acetylated histone H4. Bdf1 is phosphorylated, but the mechanism and significance of this modification have been unclear. Two distinct regions within Bdf1 are phosphorylated; one is just C terminal to the bromodomains and the other is near the C terminus. Mutational analysis shows that phosphorylation is necessary for Bdf1 function in vivo. Endogenous protein kinase CK2 purifies with Bdf1 and phosphorylates both domains. A similar mechanism may be responsible for phosphorylation of the C-terminal region of mammalian TAF1. These findings suggest that CK2 phosphorylation of Bdf1 may regulate RNA polymerase II transcription and/or chromatin structure.

Receptor-mediated regulation of the TRPM7 channel through its endogenous protein kinase domain.

TRPM7 is a ubiquitously expressed and constitutively active divalent cation-selective ion channel, whose basal activity is regulated by intracellular levels of Mg(2+) and Mg.ATP. We have investigated receptor-mediated mechanisms that may actively regulate TRPM7 activity. We here report that TRPM7 currents are suppressed by intracellular GTPgammaS, suggesting the involvement of heterotrimeric G proteins. TRPM7 currents are also inhibited by stimulating endogenous muscarinic receptors, which is mediated by G(i) because the inhibitory effect is blunted by pertussis toxin. Conversely, stimulation of endogenous G(s)-coupled beta-adrenergic receptors potentiates TRPM7 currents, whereas G(q)-coupled thrombin receptors have little effect. Consistent with the involvement of G(s)/G(i) in controlling adenylyl cyclase activity, elevations of intracellular cAMP levels enhance TRPM7 activity and prevent receptor-mediated modulation of TRPM7 activity by muscarinic and adrenergic agonists. This cAMP-dependent effect requires the functional integrity of both protein kinase A (PKA) and the endogenous kinase domain of TRPM7 because cAMP-mediated effects are abolished when treating cells with the PKA inhibitors H89 or KT5720 as well as in cells expressing phosphotransferase-deficient TRPM7 constructs. These mutant channels are also much less susceptible to GTPgammaS-mediated inhibition, suggesting that the main regulatory effect occurs through G(i)- and G(s)-mediated changes in cAMP. Taken together, our results demonstrate that TRPM7 activity is up- and down-regulated through its endogenous kinase in a cAMP- and PKA-dependent manner.

Bacterially expressed recombinant p68 RNA helicase is phosphorylated on serine, threonine, and tyrosine residues

We previously reported the expression and purification of recombinant p68 RNA helicase in a bacterial expression system. The recombinant p68 is an RNA-dependent ATPase and ATP-dependent RNA helicase. In the process of characterizing the ATPase and RNA unwinding activities of the recombinant p68, we observed that the bacterially expressed p68 RNA helicase is phosphorylated on tyrosine, serine, and threonine residues. Our data demonstrated that phosphorylations on the recombinant p68 RNA helicase affect the enzymatic activities of the protein. This is the first observation that recombinant protein expressed in bacteria Escherichia coli is phosphorylated at multiple residues by bacterial endogenous protein kinases. Our observations suggest an important mechanism in controlling the function of p68 RNA helicase by signal transduction pathways.

Protein kinase a in postmortem brain of depressed suicide victims: altered expression of specific regulatory and catalytic subunits

Background We recently reported reduced [ 3H]cyclic adenosine monophosphate binding and catalytic activity of protein kinase A in prefrontal cortex of depressed suicide victims. Here we examined the molecular basis of these alterations and whether these findings can be replicated in another cohort. Methods Prefrontal cortex from depressed suicide victims and nonpsychiatric controls were obtained from the Lenhossek Human Brain Program, Budapest and the Maryland Brain Collection Program. [ 3H]cyclic adenosine monophosphate binding and protein kinase A activity were determined by radioligand binding and enzymatic assay, respectively. Expression of catalytic and regulatory subunits was determined by quantitative reverse transcription polymerase chain reaction and Western blot, respectively. Results [ 3H]cyclic adenosine monophosphate binding and total and endogenous protein kinase A activity were significantly decreased in membrane and cytosol fractions of prefrontal cortex of depressed suicide victims from the Budapest cohort, with a similar magnitude (33%–40% reduction) as reported for the Maryland cohort. In both cohorts, selective reduction (36%–41%) in mRNA and protein expression of the regulatory RIIβ and the catalytic Cβ was observed. Conclusions Our results suggest abnormalities in [ 3H]cyclic adenosine monophosphate binding and catalytic activity kinase A in brain of depressed suicide victims, which could be due to reduced expression of RIIβ and Cβ. These abnormalities in PKA may be critical in the pathophysiology of depression.

A Chromatin Immunoprecipitation Screen Reveals Protein Kinase Cβ as a Direct RUNX1 Target Gene

RUNX1 (also known as AML1) is a DNA-binding transcription factor that functions as a tumor suppressor and developmental determinant in hematopoietic cells. Target promoters have been identified primarily through the use of differential expression strategies and candidate gene approaches but not biochemical screens. Using a chromatin immunoprecipitation screen, we identified protein kinase Cbeta as a direct RUNX1 target gene and demonstrate that endogenous RUNX1 binds the chromatinized protein kinase Cbeta promoter of U937 cells. A phylogenetically conserved RUNX1-binding site within the PKCbeta promoter binds RUNX1 in electrophoretic mobility shift analyses and confers RUNX1 responsiveness on a heterologous promoter. Changes in RUNX1 activity affect endogenous protein kinase Cbeta expression, and a dominant-negative form of RUNX1 protects U937 cells from apoptotic stimuli previously shown to be dependent on protein kinase Cbeta. This protection can be reversed by the ectopic expression of protein kinase Cbeta. Together these findings demonstrate that protein kinase Cbeta is a direct, downstream target of RUNX1 and links RUNX1 to a myeloid apoptotic pathway.

Interaction of 14-3-3 proteins with the insulin-like growth factor I receptor (IGFIR): evidence for a role of 14-3-3 proteins in IGFIR signaling

We have extended our previous yeast two-hybrid findings to show that 14-3-3β also interacts with the insulin-like growth factor I receptor (IGFIR) in mammalian cells overexpressing both proteins and that the interaction involves serine 1283 and is dependent on receptor activation. Treatment of cells with the phorbol ester PMA stimulates the interaction of 14-3-3β with the IGFIR in the absence of receptor tyrosine phosphorylation, suggesting that receptor activation leads to activation of an endogenous protein kinase that catalyzes the phosphorylation of serine 1283. To investigate the role of 14-3-3 proteins in IGF signal transduction, IGFIR structure–function studies were performed. Mutation of serine 1283 alone (S1283A) (a mutation that decreases but does not abolish the interaction of the IGFIR with 14-3-3) did not affect anchorage-independent growth of NIH 3T3 fibroblasts overexpressing the mutant receptor. However, the simultaneous mutation of this residue and the truncation of the C-terminal 27 residues of the receptor (Δ1310/S1283A) abolished the interaction of the receptor with 14-3-3 and reversed the enhanced colony formation observed with the IGFIR truncation mutation alone (Δ1310). The difference between the Δ1310 and Δ1310/S1283A transfectants in the soft agar assay was confirmed by tumorigenesis experiments. These findings suggest that 14-3-3 proteins interact with the IGFIR in vivo and that this interaction may play a role in a transformation pathway signaled by the IGFIR.

ATP-dependent regulation of SK4/IK1-like currents in rat submandibular acinar cells: possible role of cAMP-dependent protein kinase.

SK4/IK1 encodes an intermediate conductance, Ca2+ -activated K+ channel and fulfills a variety of physiological functions in excitable and nonexcitable cells. Although recent studies have provided evidence for the presence of SK4/IK1 channels in salivary acinar cells, the regulatory mechanisms and the physiological function of the channel remain unknown in these cells. Using molecular and electrophysiological techniques, we examined whether cytosolic ATP-dependent regulation of native SK4/IK1-like channel activity would involve endogenous cAMP-dependent protein kinase (PKA) in rat submandibular acinar (RSA) cells. Electrophysiological properties of tetraethylammonium (TEA) (10 mM)-insensitive, Ca2+ -dependent K+ currents in macropatches excised from RSA cells matched those of whole cell currents recorded from human embryonic kidney-293 cells heterologously expressing rat SK4/IK1 (rSK4/IK1) cloned from RSA cells. In outside-out macropatches, activity of native SK4/IK1-like channels, defined as a charybdotoxin (100 nM)-blockable current in the presence of TEA (10 mM) in the bathing solution, ran down unless both ATP and Mg2+ were present in the pipette solution. The nonhydrolyzable ATP analog AMP-PNP failed to support the channel activity as ATP did. The addition of Rp-cAMPS (10 microM), a PKA inhibitor, to the pipette solution containing ATP/Mg2+ induced a rundown of the Ca2+ -dependent K+ currents. Inclusion of cAMP (1 mM) into the pipette solution (1 microM free Ca2+) containing ATP/Mg2+ caused a gradual increase in the currents, the effect being pronounced for the currents induced by 0.1 microM free Ca2+. Forskolin (1 microM), an adenylyl cyclase activator, also increased the currents induced by 0.1 microM free Ca2+. In inside-out macropatches, cytosolic ATP/Mg2+ increased both the maximum current (proportional to the maximum channel activity) and Ca2+ sensitivity of current activation. Collectively, these results suggest that ATP-dependent regulation of native SK4/IK1-like channels, at least in part, is mediated by endogenous PKA in RSA cells.

Modeling β-Adrenergic Control of Cardiac Myocyte Contractility in Silico

The beta-adrenergic signaling pathway regulates cardiac myocyte contractility through a combination of feedforward and feedback mechanisms. We used systems analysis to investigate how the components and topology of this signaling network permit neurohormonal control of excitation-contraction coupling in the rat ventricular myocyte. A kinetic model integrating beta-adrenergic signaling with excitation-contraction coupling was formulated, and each subsystem was validated with independent biochemical and physiological measurements. Model analysis was used to investigate quantitatively the effects of specific molecular perturbations. 3-Fold overexpression of adenylyl cyclase in the model allowed an 85% higher rate of cyclic AMP synthesis than an equivalent overexpression of beta 1-adrenergic receptor, and manipulating the affinity of Gs alpha for adenylyl cyclase was a more potent regulator of cyclic AMP production. The model predicted that less than 40% of adenylyl cyclase molecules may be stimulated under maximal receptor activation, and an experimental protocol is suggested for validating this prediction. The model also predicted that the endogenous heat-stable protein kinase inhibitor may enhance basal cyclic AMP buffering by 68% and increasing the apparent Hill coefficient of protein kinase A activation from 1.0 to 2.0. Finally, phosphorylation of the L-type calcium channel and phospholamban were found sufficient to predict the dominant changes in myocyte contractility, including a 2.6x increase in systolic calcium (inotropy) and a 28% decrease in calcium half-relaxation time (lusitropy). By performing systems analysis, the consequences of molecular perturbations in the beta-adrenergic signaling network may be understood within the context of integrative cellular physiology.

Conditional protein phosphorylation regulates BK channel activity in rat cerebellar Purkinje neurons.

Large conductance calcium- and voltage-activated potassium (BK) channels are widely expressed in the mammalian central nervous system. Although the activity of BK channels in endocrine and vascular cells is regulated by protein kinases and phosphatases associated with the channel complex, direct evidence for such modulation in neurons is largely lacking. Single-channel analysis from inside-out patches isolated from the soma of dissociated rat cerebellar Purkinje neurons demonstrated that the activity of BK channels is regulated by multiple endogenous protein kinases and protein phosphatases in the membrane patch. The majority of BK channels were non-inactivating and displayed a 'low' activity phenotype determined at +40 mV and 1 muM intracellular free calcium. These channels were activated by cAMP-dependent protein kinase (PKA) associated with the patch and the extent of PKA activation was limited by an opposing endogenous type 2A-like protein phosphatase (PP2A). Importantly, PKA activation was dependent upon the prior phosphorylation status of the BK channel complex dynamically controlled by protein kinase C (PKC) and protein phosphatase 1 (PP1). In contrast, Purkinje cells also displayed a low proportion of non-inactivating BK channels with a 'high' activity under the same recording conditions and these channels were inhibited by endogenous PKA. Our data suggest that: (1) multiple endogenous protein kinases and phosphatases functionally couple to the BK channel complex to allow conditional modulation of BK channel activity in neurons, and (2) native, phenotypically distinct, neuronal BK channels are differentially sensitive to PKA-dependent phosphorylation.

Conditional protein phosphorylation regulates BK channel activity in rat cerebellar Purkinje neurons

Large conductance calcium- and voltage-activated potassium (BK) channels are widely expressed in the mammalian central nervous system. Although the activity of BK channels in endocrine and vascular cells is regulated by protein kinases and phosphatases associated with the channel complex, direct evidence for such modulation in neurons is largely lacking. Single-channel analysis from inside-out patches isolated from the soma of dissociated rat cerebellar Purkinje neurons demonstrated that the activity of BK channels is regulated by multiple endogenous protein kinases and protein phosphatases in the membrane patch. The majority of BK channels were non-inactivating and displayed a ‘low’ activity phenotype determined at +40 mV and 1 μM intracellular free calcium. These channels were activated by cAMP-dependent protein kinase (PKA) associated with the patch and the extent of PKA activation was limited by an opposing endogenous type 2A-like protein phosphatase (PP2A). Importantly, PKA activation was dependent upon the prior phosphorylation status of the BK channel complex dynamically controlled by protein kinase C (PKC) and protein phosphatase 1 (PP1). In contrast, Purkinje cells also displayed a low proportion of non-inactivating BK channels with a ‘high’ activity under the same recording conditions and these channels were inhibited by endogenous PKA. Our data suggest that: (1) multiple endogenous protein kinases and phosphatases functionally couple to the BK channel complex to allow conditional modulation of BK channel activity in neurons, and (2) native, phenotypically distinct, neuronal BK channels are differentially sensitive to PKA-dependent phosphorylation.

Pea DNA topoisomerase I is phosphorylated and stimulated by casein kinase 2 and protein kinase C.

DNA topoisomerase I catalyzes the relaxation of superhelical DNA tension and is vital for DNA metabolism; therefore, it is essential for growth and development of plants. Here, we have studied the phosphorylation-dependent regulation of topoisomerase I from pea (Pisum sativum). The purified enzyme did not show autophosphorylation but was phosphorylated in an Mg(2+)-dependent manner by endogenous protein kinases present in pea nuclear extracts. This phosphorylation was abolished with calf intestinal alkaline phosphatase and lambda phosphatase. It was also phosphorylated by exogenous casein kinase 2 (CK2), protein kinase C (PKC; from animal sources), and an endogenous pea protein, which was purified using a novel phorbol myristate acetate affinity chromatography method. All of these phosphorylations were inhibited by heparin (inhibitor of CK2) and calphostin (inhibitor of PKC), suggesting that pea topoisomerase I is a bona fide substrate for these kinases. Spermine and spermidine had no effect on the CK2-mediated phosphorylation, suggesting that it is polyamine independent. Phospho-amino acid analysis showed that only serine residues were phosphorylated, which was further confirmed using antiphosphoserine antibody. The topoisomerase I activity increased after phosphorylation with exogenous CK2 and PKC. This study shows that these kinases may contribute to the physiological regulation of DNA topoisomerase I activity and overall DNA metabolism in plants.