The Proto-oncogene PKCι regulates the alternative splicing of Bcl-x pre-mRNA

Two splice variants derived from the Bcl-x gene via alternative 5' splice site selection (5'SS) are proapoptotic Bcl-x(s) and antiapoptotic Bcl-x(L). Previously, our laboratory showed that apoptotic signaling pathways regulated the alternative 5'SS selection via protein phosphatase-1 and de novo ceramide. In this study, we examined the elusive prosurvival signaling pathways that regulate the 5'SS selection of Bcl-x pre-mRNA in cancer cells. Taking a broad-based approach by using a number of small-molecule inhibitors of various mitogenic/survival pathways, we found that only treatment of non-small cell lung cancer (NSCLC) cell lines with the phosphoinositide 3-kinase (PI3K) inhibitor LY294002 (50 μmol/L) or the pan-protein kinase C (PKC) inhibitor G?6983 (25 μmol/L) decreased the Bcl-x(L)/(s) mRNA ratio. Pan-PKC inhibitors that did not target the atypical PKCs, PKCι and PKCζ, had no effect on the Bcl-x(L)/(s) mRNA ratio. Additional studies showed that downregulation of the proto-oncogene, PKCι, in contrast to PKCζ, also resulted in a decrease in the Bcl-x(L)/(s) mRNA ratio. Furthermore, downregulation of PKCι correlated with a dramatic decrease in the expression of SAP155, an RNA trans-acting factor that regulates the 5'SS selection of Bcl-x pre-mRNA. Inhibition of the PI3K or atypical PKC pathway induced a dramatic loss of SAP155 complex formation at ceramide-responsive RNA cis-element 1. Finally, forced expression of Bcl-x(L) "rescued" the loss of cell survival induced by PKCι siRNA. In summary, the PI3K/PKCι regulates the alternative splicing of Bcl-x pre-mRNA with implications in the cell survival of NSCLC cells.     

Structure of the CaMKIIδ/Calmodulin Complex Reveals the Molecular Mechanism of CaMKII Kinase Activation

Long-term potentiation (LTP), a long-lasting enhancement in communication between neurons, is considered to be the major cellular mechanism underlying learning and memory. LTP triggers high-frequency calcium pulses that result in the activation of Calcium/Calmodulin (CaM)-dependent kinase II (CaMKII). CaMKII acts as a molecular switch because it remains active for a long time after the return to basal calcium levels, which is a unique property required for CaMKII function. Here we describe the crystal structure of the human CaMKIIδ/Ca²⁺/CaM complex, structures of all four human CaMKII catalytic domains in their autoinhibited states, as well as structures of human CaMKII oligomerization domains in their tetradecameric and physiological dodecameric states. All four autoinhibited human CaMKIIs were monomeric in the determined crystal structures but associated weakly in solution. In the CaMKIIδ/Ca²⁺/CaM complex, the inhibitory region adopted an extended conformation and interacted with an adjacent catalytic domain positioning T287 into the active site of the interacting protomer. Comparisons with autoinhibited CaMKII structures showed that binding of calmodulin leads to the rearrangement of residues in the active site to a conformation suitable for ATP binding and to the closure of the binding groove for the autoinhibitory helix by helix αD. The structural data, together with biophysical interaction studies, reveals the mechanism of CaMKII activation by calmodulin and explains many of the unique regulatory properties of these two essential signaling molecules.

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Thr90 phosphorylation of Hsp90α by protein kinase A regulates its chaperone machinery

Hsp90 (heat-shock protein 90) is one of the most important molecular chaperones in eukaryotes. Hsp90 facilitates the maturation, activation or degradation of its client proteins. It is now well accepted that both ATP binding and co-chaperone association are involved in regulating the Hsp90 chaperone machinery. However, other factors such as post-translational modifications are becoming increasingly recognized as being involved in this process. Recent studies have reported that phosphorylation of Hsp90 plays an unanticipated role in this process. In the present study, we systematically investigated the impact of phosphorylation of a single residue (Thr90) of Hsp90α (pThr90-Hsp90α) on its chaperone machinery. We demonstrate that protein kinase A specifically phosphorylates Hsp90α at Thr90, and that the pThr9090-Hsp90α level is significantly elevated in proliferating cells. Thr90 phosphorylation affects the binding affinity of Hsp90α to ATP. Subsequent examination of the interactions of Hsp90α with co-chaperones reveals that Thr90 phosphorylation specifically regulates the association of a subset of co-chaperones with Hsp90α. The Hsp90α T90E phosphor-mimic mutant exhibits increased association with Aha1 (activator of Hsp90 ATPase homologue 1), p23, PP5 (protein phosphatase 5) and CHIP (C-terminus of Hsp70-interacting protein), and decreased binding affinity with Hsp70, Cdc37 (cell division cycle 37) and Hop [Hsc70 (heat-shock cognate protein 70)/Hsp90-organizing protein], whereas its interaction with FKBP52 (FK506-binding protein 4) is only moderately affected. Moreover, we find that the ability of the T90E mutant to form complexes with its clients, such as Src, Akt or PKCγ (protein kinase Cγ), is dramatically impaired, suggesting that phosphorylation affects its chaperoning activity. Taken together, the results of the present study demonstrate that Thr90 phosphorylation is actively engaged in the regulation of the Hsp90α chaperone machinery and should be a generic determinant for the cycling of Hsp90α chaperone function.     

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.     

Realizing the allosteric potential of the tetrameric protein kinase A RIα holoenzyme

PKA holoenzymes containing two catalytic (C) subunits and a regulatory (R) subunit dimer are activated cooperatively by cAMP. While cooperativity involves the two tandem cAMP binding domains in each R-subunit, additional cooperativity is associated with the tetramer. Of critical importance is the flexible linker in R that contains an inhibitor site (IS). While the IS becomes ordered in the R:C heterodimer, the overall conformation of the tetramer is mediated largely by the N-Linker that connects the?D/D domain to the IS. To understand how the N-Linker contributes to assembly of tetrameric holoenzymes, we engineered a monomeric RIα that contains most of the N-Linker, RIα(73-244), and crystallized a holoenzyme complex. Part of the N-linker is now ordered by interactions with a symmetry-related dimer. This complex of two symmetry-related dimers forms a tetramer that reveals novel mechanisms for allosteric regulation and has many features associated with full-length holoenzyme. A model of the tetrameric holoenzyme, based on this structure, is consistent with previous small angle X-ray and neutron scattering data, and is validated with new SAXS data and with an RIα mutation localized to a novel interface unique to the tetramer.     

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.     

Diacylglycerol kinase θ counteracts protein kinase C-mediated inactivation of the EGF receptor

Epidermal growth factor receptor (EGFR) activation is negatively regulated by protein kinase C (PKC) signaling. Stimulation of A431 cells with EGF, bradykinin or UTP increased EGFR phosphorylation at Thr654 in a PKC-dependent manner. Inhibition of PKC signaling enhanced EGFR activation, as assessed by increased phosphorylation of Tyr845 and Tyr1068 residues of the EGFR. Diacylglycerol is a physiological activator of PKC that can be removed by diacylglycerol kinase (DGK) activity. We found, in A431 and HEK293 cells, that the DGKθ isozyme translocated from the cytosol to the plasma membrane, where it co-localized with the EGFR and subsequently moved into EGFR-containing intracellular vesicles. This translocation was dependent on both activation of EGFR and PKC signaling. Furthermore, DGKθ physically interacted with the EGFR and became tyrosine-phosphorylated upon EGFR stimulation. Overexpression of DGKθ attenuated the bradykinin-stimulated, PKC-mediated EGFR phosphorylation at Thr654, and enhanced the phosphorylation at Tyr845 and Tyr1068. SiRNA-induced DGKθ downregulation enhanced this PKC-mediated Thr654 phosphorylation. Our data indicate that DGKθ translocation and activity is regulated by the concerted activity of EGFR and PKC and that DGKθ attenuates PKC-mediated Thr654 phosphorylation that is linked to desensitisation of EGFR signaling.     

Is alternative splicing of mammalian genes conservative?

The conservation of alternative splicing in orthologous genes from the human and mouse genomes was analyzed. Alternatively spliced mouse genes from the AsMamDB database were used to scan the draft human genome. The mouse protein isoforms were aligned with respect to orthologous human genes, and thus the exon-intron structure of the latter was established. Proteins isoforms that could not be aligned throughout their length were analyzed in detail using the human EST alignment.     

Tumor microenvironment–associated modifications of alternative splicing

Pre-mRNA alternative splicing is modified in cancer, but the origin and specificity of these changes remain unclear. Here, we probed ovarian tumors to identify cancer-associated splicing isoforms and define the mechanism by which splicing is modified in cancer cells. Using high-throughput quantitative PCR, we monitored the expression of splice variants in laser-dissected tissues from ovarian tumors. Surprisingly, changes in alternative splicing were not limited to the tumor tissues but were also found in the tumor microenvironment. Changes in the tumor-associated splicing events were found to be regulated by splicing factors that are differentially expressed in cancer tissues. Overall, ∼20% of the alternative splicing events affected by the down-regulation of the splicing factors QKI and RBFOX2 were altered in the microenvironment of ovarian tumors. Together, our results indicate that the tumor microenvironment undergoes specific changes in alternative splicing orchestrated by a limited number of splicing factors.     

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.     

Extensive regulation of NAGNAG alternative splicing: new tricks for the spliceosome?

A recent study using massive parallel sequencing demonstrates unequivocally that alternative tandem acceptor splicing is tissue-specifically regulated.     

Limited proteolysis of the catalytic subunit of cAMP-dependent protein kinase — A membranal regulatory device?

Brush border membranes isolated from rat small intestine were found to possess a cAMP-dependent protein kinase activity. Upon addition of cAMP, a rapid, time-dependent inactivation of this enzyme occurs, which was found to be due to a proteolytic activity identified in the membranes. This activity could not be assigned to previously known brush border proteases. The inactivation and the proteolytic degradation of the kinase could be reproduced also with the pure catalytic subunit of cAMP-dependent protein kinase (C) from rabbit skeletal muscle (M.W. 40000) which was cleaved by the membranal proteolytic activity with concomitant quantitative appearance of a degradation product (M.W. 30000) devoid of kinase activity. The membranal proteolytic activity appears to be specific for C since: (1) it does not degrade the other endogenous proteins in the membrane preparation; (2) it does not degrade any of six arbitrarily chosen proteins from other sources; (3) it catalyzes a limited proteolysis of C which could not be simulated by other proteolytic enzymes such as trypsin, clostripain, chymotrypsin and papain. The attack of C by the membranal protease is blocked by the presence of the nucleotide substrate of the kinase (MgATP). In addition, the undissociated and inactive form of the enzyme (R₂C₂) does not lose its potential enzymatic activity, and neither its catalytic nor its regulatory subunits are digested by the protease. The specific, restricted and limited action of the protease, together with the prevention of its action by the substrate and the regulatory protein (R) of the kinase raise the possibility that the membranal protease may have a distinct physiological (possibly regulatory) assignment.     

β-N-oxalyl-L-α,β-diaminopropionic acid regulates mitogen-activated protein kinase signaling by down-regulation of phosphatidylethanolamine-binding protein 1

β-N-Oxalyl-L-α,β-diaminopropionic acid (l-ODAP) an α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor agonist activates protein kinase C in white leghorn chick brain. The current study focuses on the protein kinase C downstream signaling targets associated with L-ODAP excitotoxicity in SK-N-MC human neuroblastoma cells and white leghorn male chick (Gallus domesticus) brain extracts. L-ODAP treatment in SK-N-MC cells (1.5 mM) and chicks (0.5 mg/g body weight) results in a decreased expression and increased phosphorylation of phosphatidylehthanolamine-binding protein 1 (PEBP1) up to 4 h which however, returns to normal by 8 h. D-ODAP, the non-toxic enantiomer however, did not affect PEBP1 levels in either chick brain or SK-N-MC cells. Decreased PEBP1 expression correlated with subsequent activation of Raf-1, MEK and ERK signaling components of the mitogen-activated protein kinase cascade and nuclear translocation of hypoxia inducible factor-1α (HIF-1α) in chick brain nuclear extracts and SK-N-MC cells. SK-N-MC cells over-expressing PEBP1 inhibited nuclear translocation of HIF-1α when treated with l-ODAP, indicating that down-regulation of PEBP1 is responsible for HIF-1α stabilization and nuclear localization. Excitotoxicity of L-ODAP may thus be the result of phosphorylation and down-regulation of PEBP1, a crucial signaling protein regulating diverse signaling cascades. L-ODAP induced convulsions and seizures in chicks could be the result of a hypoxic insult to brain.