Treatment of Jurkat cells with specific inhibitors of protein kinase CK2 induces apoptosis. Here we provide evidence that the antiapoptotic effect of CK2 can be at least partially mediated by upregulation of the Akt/PKB pathway. Such a conclusion is based on the following observations: (1) inhibition of CK2 by cell treatment with two structurally unrelated CK2 inhibitors induces downregulation of Akt/PKB, as judged from decreased phosphorylation of its physiological targets, and immunoprecipitate kinase assay; (2) similar results are observed upon reduction of CK2 catalytic subunit by the RNA-interference technique; (3) Akt/PKB Ser129 is phosphorylated by CK2 in vitro and in vivo; (4) such a phosphorylation of activated Akt/PKB correlates with a further increase in catalytic activity. These data disclose an unanticipated mechanism by which constitutive phosphorylation by CK2 may be required for maximal activation of Akt/PKB.
Type II hexokinase is overexpressed in most neoplastic cells, and it mainly localizes on the outer mitochondrial membrane. Hexokinase II dissociation from mitochondria triggers apoptosis. The prevailing model postulates that hexokinase II release from its mitochondrial interactor, the voltage-dependent anion channel, prompts outer mitochondrial membrane permeabilization and the ensuing release of apoptogenic proteins, and that these events are inhibited by growth factor signalling. Here we show that a hexokinase II N-terminal peptide selectively detaches hexokinase II from mitochondria and activates apoptosis. These events are abrogated by inhibiting two established permeability transition pore modulators, the adenine nucleotide translocator or cyclophilin D, or in cyclophilin D knock-out cells. Conversely, insulin stimulation or genetic ablation of the voltage-dependent anion channel do not affect cell death induction by the hexokinase II peptide. Therefore, hexokinase II detachment from mitochondria transduces a permeability transition pore opening signal that results in cell death and does not require the voltage-dependent anion channel. These findings have profound implications for our understanding of the pathways of outer mitochondrial membrane permeabilization and their inactivation in tumors.
Protein kinase casein kinase 1 (CK1) phosphorylates Ser-45 of -catenin, ''priming'' the subsequent phosphorylation by glycogen synthase-3 of residues 41, 37, and 33. This concerted phosphorylation of -catenin signals its degradation and prevents its function in triggering cell division. The sequence around Ser-45 does not conform to the canonical consensus for CK1 substrates, which prescribes either phosphoamino acids or acidic residues in position n؊3 from the target serine. However, the -catenin sequence downstream from Ser-45 is very similar to a sequence recognized by CK1 in nuclear factor for activated T cells 4. The common features include an SLS motif followed two to five residues downstream by a cluster of acidic residues. Synthetic peptides reproducing residues 38 -65 of -catenin were assayed with purified rat liver CK1 or recombinant CK1␣ and CK1␣L from zebrafish. The results demonstrate that SLS and acidic cluster motifs are crucial for CK1 recognition. Pro-44 and Pro-52 are also important for efficient phosphorylation. Similar results were obtained with the different isoforms of CK1. Phosphorylation of mutants of full-length recombinant -catenin from zebrafish confirmed the importance of the SLS and acidic cluster motifs. A search for proteins with similar motifs yielded, among other proteins, adenomatous polyposis coli, previously found to be phosphorylated by CK1. There is a strong correlation of -catenin mutations found in thyroid tumors with the motifs recognized by CK1 in this protein.consensus sequence ͉ nuclear factor for activated T cells 4 ͉ adenomatous polyposis coli ͉ Wnt signaling ͉ thyroid tumor mutations
In addition to tyrosine sites, FAK (focal adhesion kinase) is phosphorylated on multiple serine residues. In the present study, the regulation of two of these sites, Ser-722 (S1) and Ser-911 (S4), was investigated. Phosphorylation of S1 (but not S4) decreased in resuspended cells, and recovered during spreading on fibronectin, indicating adhesion-dependent regulation. GSK3 (glycogen synthase kinase 3) inhibitors decreased S1 phosphorylation, and siRNA (short interfering RNA) silencing indicated further the involvement of GSK3beta. Furthermore, GSK3beta was found to become activated during cell spreading on fibronectin, and to physically associate with FAK. S1 phosphorylation was observed to decrease in wounded cell monolayers, while GSK3beta underwent inactivation and later was observed to increase to the original level within 24 h. Direct phosphorylation of S1, requiring pre-phosphorylation of Ser-726 in the +4 position, was demonstrated using purified GSK3 and a synthetic peptide containing FAK residues 714-730. An inhibitory role for S1 phosphorylation in FAK signalling was indicated by findings that both alanine substitution for S1 and dephosphorylation of S1 by PP1 (serine/threonine protein phosphatase type-1) resulted in an increase in FAK kinase activity; likewise, this role was also shown by cell treatment with the GSK3 inhibitor LiCl. The inhibitory role was confirmed by the finding that cells expressing FAK with alanine substitution for S1 displayed improved cell spreading and faster migration in wound-healing and trans-well assays. Finally, the finding that S1 phosphorylation increased in cells treated with the PP1 inhibitor okadaic acid indicated targeting of this site by PP1. These results indicate an additional mechanism for regulation of FAK activity during cell spreading and migration, involving Ser-722 phosphorylation modulated through the competing actions of GSK3beta and PP1.
AurA (Aurora-A) is a ubiquitous protein kinase regulating entry into mitosis and shown to promote transformation upon overexpression. In order to gain information on the structural features determining its substrate specificity, we assayed human recombinant AurA on a variety of phosphoacceptor peptide substrates including a series of properly modified derivatives of the Kemptide (ALRRASLGAA). The data presented here show that AurA is a basophilic Ser/Thr protein kinase recognizing the consensus R/K/N-R-X-S/T-B, where B denotes any hydrophobic residue with the exception of Pro. We show that the presence of a Pro at position n+1 fully abrogates phosphorylation of the peptide substrate. Although the consensus for AurA is reminiscent of that of PKA (protein kinase A), it significantly differs from the latter for a much more stringent dependence on the hydrophobic residue at n+1 and for its tolerance of residues other than Arg at position n-3. Based on the finding that the peptide ALKRASLGAA is not a substrate of PKA while still providing a sensitive assay of AurA activity, we suggest that this peptide may be used for differential screening of the two kinases. We have further validated the AurA consensus by generating a peptide (APSSRRTT288LCGT) that comprises the main AurA autophosphorylation site and by showing that AurA phosphorylated this peptide exclusively at one site fulfilling its consensus (Thr288). Moreover, we show that AurA could autophosphorylate at Thr288 through an intermolecular mechanism of reaction and that, in vivo, PKA was not involved with Thr288 phosphorylation. The evidence obtained in the present study provides a rational tool for predicting AurA sites in potential substrates of physiological significance.
Recombinant human a subunit from casein kinase-2 (CK-2) was subjected, either alone or in combination with recombinant human fi subunit, to high temperature, tryptic digestion and urea treatment. In all three cases, it was shown that the presence of the fi subunit could drastically reduce the loss of kinase activity, strongly suggesting a protective function for the fi subunit. Assaying different peptides for specificity toward the recombinant a subunit and the recombinant reconstituted enzyme, showed that the presence of the fi subunit could modify the specificity of the catalytic a subunit. Therefore, a dual function for the p subunit is proposed which confers both specificity and stability to the catalytic a subunit within the CK-2 holoenzyme complex. The peptide DLEPDEELEDNPNQSDL, reproducing the highly acidic amino acid 55 -71 segment of the human fi subunit, counteracts the stimulatory effect of the p subunit on the a subunit activity and partially substitutes the fi subunit in conferring thermal stability to the a subunit. No such effect is induced by the peptide MSSSEEVSW, reproducing the N-terminal segment of the fl subunit including the autophosphorylation site. It is suggested that the acidic domain of the fi subunit, encompassing residues 55 -71, plays a role in the interactions between the fi and a subunits.Since the first isolation and sequencing of cDNA species from casein kinase-2 (CK-2) a and p subunits from Drosophilu melanogaster by Saxena et al.[l], several other cDNA species of CK-2 subunits from yeast to man have been isolated and sequenced [2 -9). Subsequently, attempts were made to express ci cDNA in a bacterial expression system [lo-121. Recently, the expression of both a and p cDNA in Escherichia coli was reported [12]. The availability of both recombinant subunits in large quantities and of synthetic peptides reproducing characteristic segments of the s subunit allowed, for the first time, extensive biochemical and biophysical studies on the stability and specificity of the individual subunits and the reconstituted recombinant holoenzyme. We have focused our attention primarily on the influence of the fi subunit on the high temperature, proteolysis and urea sensitivity of the LX subunit, as well as on its peptide substrate specificity which is shown here to be significantly different from that of native and reconstituted CK-2. MATERIALS AND METHODSCloning and expression of CK-2 a and / 3 subunits The cloning and expression of the subunits was carried out as described earlier [I21 using the pT7-7 derived vectors pBBCorrespondence to
Human T-cell leukemia virus type 1 encodes a number of "accessory" proteins of unclear function; one of these proteins, p13 II , is targeted to mitochondria and disrupts mitochondrial morphology. The present study was undertaken to unravel the function of p13 II through (i) determination of its submitochondrial localization and sequences required to alter mitochondrial morphology and (ii) an assessment of the biophysical and biological properties of synthetic peptides spanning residues 9 -41 (p13 9 -41 ), which include the amphipathic mitochondrialtargeting sequence of the protein. p13 9 -41 folded into an ␣ helix in micellar environments. Fractionation and immunogold labeling indicated that full-length p13 II accumulates in the inner mitochondrial membrane. p13 9 -41 induced energy-dependent swelling of isolated mitochondria by increasing inner membrane permeability to small cations (Na ؉ , K ؉ ) and released Ca 2؉ from Ca 2؉ -preloaded mitochondria. These effects as well as the ability of full-length p13 II to alter mitochondrial morphology in cells required the presence of four arginines, forming the charged face of the targeting signal. The mitochondrial effects of p13 9 -41 were insensitive to cyclosporin A, suggesting that full-length p13 II might alter mitochondrial permeability through a permeability transition pore-independent mechanism, thus distinguishing it from the mitochondrial proteins Vpr and X of human immunodeficiency virus type 1 and hepatitis B virus, respectively.HTLV-1 1 is a complex retrovirus that is associated with two distinct pathologies, a leukemia/lymphoma of mature CD4 ϩ
By using a P-casein-derived specific peptide substrate for mammary gland Golgi-enriched-fraction casein kinase, phosphorylating activity has been detected in the Golgi apparatus of rat liver, spleen and to a lesser extent, kidney and brain, while the other post-nuclear cytoplasmic fractions are totally devoid of such a casein kinase activity. In contrast ubiquitous protein kinases CKI and CK2 (casein kinases 1 and 2), tested with their specific peptide substrates, display different subcellular distribution and are almost undetectable in the Golgi fraction. The absence of CK2 in the Golgi fraction has been also confirmed using specific antibodies. The relatedness between the liver Golgi apparatus casein kinase (G-CK) and the bona fide mammary gland Golgi-enriched-fraction casein kinase (GEF-CK) is supported by a variety of observations, notably : (a) identical peptide substrate specificity, consistent with an S-X-E-X consensus sequence; (b) preference for MnZ+, and, to a lesser extent, Co2+, over ME", as activating cation ; (c) superimposable elution profiles from DEAE-Sepharose, heparin-Sepharose, and Superdex 200, this latter consistent with a molecular mass around 500 kDa; (d) insensitivity to staurosporine and heparin (a potent inhibitor of CK2) and inability to use GTP as phosphate donor (by contrast to CK2). These data provide the evidence for the existence of a third class of ubiquitous casein kinases here termed G-CK, distinct from CK1 and CK2, specifically located to the Golgi apparatus and related to the bonafide casein kinase(s) responsible for the phosphorylation of casein secreted from lactating mammary gland. The possible involvement of G-CK in the phosphorylation of secretory pathways proteins at S-X-E motifs is discussed.
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