Protein kinase CK1 from Trypanosoma cruzi

A protein kinase activity, which uses casein as a substrate, has been purified to homogeneity from the epimastigote stage of Trypanosoma cruzi, by sequential chromatography on Q sepharose, heparin sepharose, phenyl sepharose, and alpha-casein agarose. An apparent molecular weight of 36,000 was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel filtration chromatography and sedimentation analyses demonstrated that the purified native enzyme is a monomer with a sedimentation coefficient of 2.9 S. The hydrodynamic parameters indicated that the shape of the protein is globular with a frictional ratio f/f(o) = 1.36 and a Stokes radius of 27.7 A. When two selective peptide substrates for protein kinases CK1 and CK2 were used (RRKDLHDDEEDEAM. SITA and RRRADDSDDDDD, respectively), the purified kinase was shown to predominantly phosphorylate the CK1-specific peptide. Additionally, the enzyme was inhibited by N-(2-amino-ethyl)-5-chloroisoquinoline-8-sulfonamide, a specific inactivator of CK1s from mammals. Based on these results, we concluded that the purified kinase corresponds to a parasite CK1.     

Carboxy-terminal phosphorylation of SIRT1 by protein kinase CK2

Previous analyses of the sirtuin family of histone deacetylases and its most prominent member SIRT1 have focused primarily on the identification of cellular targets exploring the underlying molecular mechanisms of its implicated function in the control of metabolic homeostasis, differentiation, apoptosis and cell survival. So far, little is known about the regulation of SIRT1 itself. In the study presented herein, we assigned the main region of SIRT1 in vivo phosphorylation to amino acids 643-691 of the unique carboxy-terminal domain. Furthermore, we demonstrate that SIRT1 is a substrate for protein kinase CK2 both in vitro and in vivo. Both, deletion construct analyses and serine-to-alanine mutations identified SIRT1 Ser-659 and Ser-661 as major CK2 phosphorylation sites that are phosphorylated in vivo as well.     

Phosphorylation of the regulatory beta-subunit of protein kinase CK2 by checkpoint kinase Chk1: identification of the in vitro CK2beta phosphorylation site

The regulatory beta-subunit of protein kinase CK2 mediates the formation of the CK2 tetrameric form and it has functions independent of CK2 catalytic subunit through interaction with several intracellular proteins. Recently, we have shown that CK2beta associates with the human checkpoint kinase Chk1. In this study, we show that Chk1 specifically phosphorylates in vitro the regulatory beta-subunit of CK2. Chymotryptic peptides and mutational analyses have revealed that CK2beta is phosphorylated at Thr213. Formation of a stable complex between CK2beta and Chk1 is not affected by the modification of Thr213 but it does require the presence of an active Chk1 kinase.     

The human IKKbeta subunit kinase domain displays CK2-like phosphorylation specificity

NF-κB activation in response to pro-inflammatory stimuli relies upon phosphorylation of IκBα at serines 32 and 36 by the β subunit of the IκB kinase complex (IKK). In this study, we build upon the observation that highly purified human IKKβ subunit preparations retain this specificity in vitro. We show that IKKβ constructs that lack their carboxy-terminus beginning at the leucine zipper motif fail to phosphorylate IκBα at Ser-32 and Ser-36. Rather, these constructs, which contain the entire IKKβ subunit kinase domain, phosphorylate serine and threonine residues contained within the IκBα carboxy-terminal PEST region. Furthermore, removal of the leucine zipper and helix-loop-helix regions converts IKKβ to monomer. We propose that the helix-loop-helix of the human IKKβ subunit is necessary for restricting substrate specificity toward Ser-32 and Ser-36 in IκBα and that in the absence of its carboxy-terminal protein structural motifs the human IKKβ subunit kinase domain exhibits a CK2-like phosphorylation specificity.     

Specific binding of protein kinase CK2 catalytic subunits to tubulin

Protein kinase CK2 is composed of two regulatory beta-subunits and two catalytic alpha- or alpha'-subunits. To analyse these subunits individually we generated antibodies against unique peptides derived from the alpha-, alpha'- and beta-subunit. Immunofluorescence studies with these antibodies revealed the presence of all three CK2 subunits in the cytoplasm and weakly in the nucleus with strong signals around the nuclear membrane. Double staining experiments revealed a co-localisation of all three subunits with tubulin. A direct association between the CK2 alpha- and the alpha'-subunit and tubulin was confirmed by co-immunoprecipitation experiments as well as by Far Western analysis. There was no binding of the CK2 beta-subunit to tubulin. Thus, with tubulin we have identified a new binding partner specific for the catalytic subunits of CK2.     

The tubulin genes of Trypanosoma cruzi

The organization of the alpha- and beta-tubulin genes in the genome of Trypanosoma cruzi have been analysed by Southern blotting using tubulin probes derived from Trypanosoma brucei. The tubulin array appears to be more complex in this organism than in other members of the same family. Some tubulin genes are tightly clustered in an alternating (alpha-beta)n array with a basic repeat unit length of 4.3 kb. However, other pairs of alternating alpha- and beta-tubulin sequences appear to be physically separated from the basic group. This finding indicates that the tubulin gene cluster present in T. cruzi is less perfectly conserved than in T. brucei. T. (Herpetosoma) rangeli is similar to T. (Schizotrypanum) cruzi in its tubulin gene organization whereas most of these genes are tandemly clustered in the genome of T. (Trypanozoon) evansi, with a basic repeat unit length of 3.6 kb as previously described for T. (Trypanozoon) brucei. Two overlapping recombinant clones containing T. cruzi tubulin sequences have been isolated from a genomic cosmid library of T. cruzi epimastigotes using the T. brucei tubulin probes. Partial sequencing of the T. cruzi beta-tubulin gene has confirmed its identity and shows more than 70% homology with the sea urchin, chicken and T. b. rhodesiense beta-tubulin reported gene sequences. Analysis of tubulin gene organization through the parasite life cycle does not show evidence of major rearrangements within the repeat unit. Several T. cruzi strains and cloned lines whilst sharing the 4.3-kb tubulin repeat unit, exhibited very variable tubulin gene organization with tubulin probes. These striking differences in the organization of this structural gene among T. cruzi strains and cloned lines suggest that the heterogeneity previously reported in parasite populations may be related to a very dynamic, diploid genome.     

Divalent cation hinder the solubilization of a tubulin kinase activity from Trypanosoma cruzi epimastigotes

Trypanosoma cruzi epimastigotes were extracted under various conditions in order to examine the role of divalent cations in the solubilization of microtubule proteins. When epimastigotes were homogenized in the presence of 5 mM Mg+2 and 5 mM Ca+2, a protein kinase responsible for phosphorylating tubulin, as well as the tubulin that became phosphorylated, remained tightly associated with the parasite particulate and detergent-resistant fractions. On the contrary, tubulin kinase and its substrate were predominantly released into the parasite cytosolic and detergent-soluble fractions, when epimastigotes were extracted in the presence of 5 mM EDTA and 5 mM EGTA. These evidences demonstrated a divalent cation-dependent solubilization of the enzyme responsible for the phosphorylation of tubulin in T. cruzi epimastigotes and suggested a tight association between tubulin and this kinase. Under all conditions tested, tubulin kinase activity in epimastigote extracts was lower than the addition of the corresponding value in the parasite cytosolic and membranous fractions, suggesting the presence of a kinase inhibitor or regulatory subunit which also seemed to be modulated by divalent cations. Additionally, inhibition experiments in the presence of heparin, 2,3-bisphosphoglycerate and GTP established that the parasite tubulin kinase corresponded to a protein kinase CK2.     

Molecular cloning and characterization of mitogen-activated protein kinase 2 in Trypanosoma cruzi

Mitogen-activated protein kinase (MAPK) pathways are major signal transduction systems by which eukaryotic cells convert environmental cues to intracellular events such as proliferation and differentiation. We have identified a Trypanosoma cruzi homologue of the MAPK family that we have called TcMAPK2. Sequence analyses demonstrates TcMAPK2 has high homology with lower eukaryotic ERK2 but has significant differences from mammalian ERK2. Enzymatic assays of both recombinant TcMAPK2 and native protein obtained by immunoprecipitation using anti-TcMAPK2 demonstrated that both preparations of TcMAPK2 were catalytically active. Immunofluorescence analysis of the subcellular localization of TcMAPK2 determined it is mainly cytoplasmic in epimastigotes, along the flagella in trypomastigotes and on the plasma membrane of intracellular amastigotes. Phosphorylated TcMAPK2 was highest in trypomastigotes and lowest in amastigotes. Recombinant TcMAPK2 was able to phosphorylate the recombinant protein of a cAMP specific phosphodiesterase. Overexpression of TcMAPK2 in epimastigotes inhibited growth and development leading to death. TcMAPK2 has an important role in the stress response of the parasite and may be important in regulating proliferation and differentiation.Key words: Trypanosoma cruzi, mitogen-activated protein kinase, phosphorylation     

Molecular cloning and characterization of mitogen-activated protein kinase 2 in Trypanosoma cruzi

Mitogen-activated protein kinase (MAPK) pathways are major signal transduction systems by which eukaryotic cells convert environmental cues to intracellular events such as proliferation and differentiation. We have identified a Trypanosoma cruzi homologue of the MAPK family that we have called TcMAPK2. Sequence analyses demonstrates TcMAPK2 has high homology with lower eukaryotic ERK2 but has significant differences from mammalian ERK2. Enzymatic assays of both recombinant TcMAPK2 and native protein obtained by immunoprecipitation using anti-TcMAPK2 demonstrated that both preparations of TcMAPK2 were catalytically active. Immunofluorescence analysis of the subcellular localization of TcMAPK2 determined it is mainly cytoplasmic in epimastigotes, along the flagella in trypomastigotes and on the plasma membrane of intracellular amastigotes. Phosphorylated TcMAPK2 was highest in trypomastigotes and lowest in amastigotes. Recombinant TcMAPK2 was able to phosphorylate the recombinant protein of a cAMP specific phosphodiesterase. Over-expression of TcMAPK2 in epimastigotes inhibited growth and development leading to death. TcMAPK2 has an important role in the stress response of the parasite and may be important in regulating proliferation and differentiation.     

Cyclic AMP-dependent protein kinase activity in Trypanosoma cruzi.

A cyclic AMP-dependent protein kinase activity from epimastigote forms of Trypanosoma cruzi was characterized. Cytosolic extracts were chromatographed on DEAE-cellulose columns, giving two peaks of kinase activity, which were eluted at 0.15 M- and 0.32 M-NaCl respectively. The second activity peak was stimulated by nanomolar concentrations of cyclic AMP. In addition, a cyclic AMP-binding protein co-eluted with the second kinase activity peak. Cyclic AMP-dependent protein kinase activity was further purified by gel filtration, affinity chromatography on histone-agarose and cyclic AMP-agarose, as well as by chromatography on CM-Sephadex. The enzyme ('holoenzyme') could be partially dissociated into two different components: 'catalytic' and 'regulatory'. The 'regulatory' component had specific binding for cyclic AMP, and it inhibited phosphotransferase activity of the homologous 'catalytic component' or of the 'catalytic subunit' from bovine heart. Cyclic AMP reversed these inhibitions. A 'holoenzyme preparation' was phosphorylated in the absence of exogenous phosphate acceptor and analysed by polyacrylamide-gel electrophoresis. A 56 kDa band was phosphorylated. The same preparation was analysed by Western blotting, by using polyclonal antibodies to the regulatory subunits of protein kinases type I or II. Both antibodies reacted with the 56 kDa band.     

Xenopus laevis occludin. Identification of in vitro phosphorylation sites by protein kinase CK2 and association with cingulin.

Occludin is a protein component of the membrane domain of tight junctions, and has been shown to be phosphorylated in vivo in cultured cells and Xenopus laevis embryos. However, nothing is known about the identity of specific occludin kinase(s) and occludin phosphorylation site(s). Furthermore, nothing is known about the interaction of occludin with cingulin, a cytoplasmic plaque component of tight junctions. Here we report the isolation and sequencing of a complete X. laevis occludin cDNA, and experiments aimed at mapping X. laevis occludin in vitro phosphorylation site(s) and characterizing occludin interaction with cingulin. The sequence of Xenopus occludin is homologous to that of occludins from other species, with identities ranging from 41% to 58%. Bacterially expressed domain E of Xenopus occludin (amino acids 247-493) was a good substrate for protein kinase CK2 (stoichiometry 10.8%, Km 8.4 microM) but not for CK1 kinase, protein kinase A, cdc2 kinase, MAP kinase or syk kinase. Residues Thr375 and Ser379 were identified as potential CK2 phosphorylation sites in this region based on sequence analysis. Mutation of Ser379 to aspartic acid or alanine reduced phosphorylation by CK2 by approximately 50%, and double mutation of Ser379 into aspartic acid and Thr375 into aspartic acid essentially abolished phosphorylation. Glutathione S-transferase (GST) pull-down experiments using extracts of Xenopus A6 epithelial cells showed that constructs of GST fused to wild-type and mutant forms of the C-terminal region of X. laevis occludin associate with several polypeptides, and immunoblot analysis showed that one of these polypeptides is cingulin. GST pull-down experiments using in vitro translated, full-length Xenopus cingulin indicated that cingulin interacts directly with the C-terminal region of occludin.     

Inhibition of protein kinase activity from Trypanosoma cruzi and Trypanosoma gambiense by 3'-deoxyadenosine.

3'-Deoxydadenosine was found to be a potent inhibitor of nucleoside-stimulated protein kinase activity from culture forms of Trypanosoma cruzi and bloodstream forms of Trypanosoma gambiense. The type of inhibition by 3'-deoxyadenosine was competitive with respect to ATP. The inhibition constants for 3'-deoxyadenosine were determined to be 0.11mM and 0.085mM for the enzyme from T. cruzi and T. gambiense, respectively. The apparent Km value for ATP was 0.2mM for both enzymes. 2'-Deoxyadenosine was less effective as inhibitor of the protein kinase activity. The inhibition constants were calculated to be 0.8mM and 0.67mM, respectively.     

High phosphorylation of HBV core protein by two alpha-type CK2-activated cAMP-dependent protein kinases in vitro

Two alpha-type CK2-activated PKAs (CK2-aPKAIalpha and CK2-aPKAIIalpha) were biochemically characterized in vitro using GST-HBV core fusion protein (GST-Hcore) and GST-Hcore157B as phosphate acceptors. It was found that (i), in the absence of cAMP, these two CK2-aPKAs phosphorylated both Ser-170 and Ser-178 on GST-Hcore and Hcore157B; (ii) this phosphorylation was approx. 4-fold higher than their phosphorylation by cAMP-activated PKAs; and (iii) suramin effectively inhibited the phosphorylation of Hcore157B by CK2-aPKAIIalpha through its direct binding to Hcore157B in vitro. These results suggest that high phosphorylation of HBV-CP by two CK2-aPKAs, in the absence of cAMP, may be involved in the pregenomic RNA (pgRNA) encapsidation and DNA-replication in HBV-infected cells.     

Unique features of HIV-1 Rev protein phosphorylation by protein kinase CK2 ('casein kinase-2')

The HIV-1 Rev transactivator is phosphorylated in vitro by protein kinase CK2 at two residues, Ser-5 and Ser-8; these sites are also phosphorylated in vivo. Here we show that the mechanism by which CK2 phosphorylates Rev is unique in several respects, notably: (i) it is fully dependent on the regulatory, beta-subunit of CK2; (ii) it relies on the integrity of an acidic stretch of CK2 beta which down-regulates the phosphorylation of other substrates; (iii) it is inhibited in a dose-dependent manner by polyamines and other polycationic effectors that normally stimulate CK2 activity. In contrast, a peptide corresponding to the amino-terminal 26 amino acids of Rev, including the phosphoacceptor site, is readily phosphorylated by the catalytic subunit of CK2 even in the absence of the beta-subunit. These data, in conjunction with the observation that two functionally inactive derivatives of Rev with mutations in its helix-loop-helix motif are refractory to phosphorylation, indicate the phosphorylation of Rev by CK2 relies on conformational features of distinct regions that are also required for the transactivator's biological activity.