Protein kinase CK2 phosphorylates BAD at threonine-117

Reversible phosphorylation of the 22 kDa BAD protein is crucial for cell survival. Five phosphorylation sites, all serines, had been identified. Here we report on number six. It is threonine-117 phosphorylated by the constitutively active kinase, CK2. Phosphoamino acid analysis and phospho-specific antibodies confirmed Thr117 as additional phosphorylation site. Immunoprecipitation furthermore revealed that BAD is phosphorylated at Thr117 in cultured cortical neurons. PP1, PP2A and PP2C dephosphorylated BAD at Thr117, but PP2B did not. The discovery of the constitutively active CK2 phosphorylating BAD is shedding an unexpected light in the otherwise strictly signal-regulated phosphorylation events on BAD.     

Novel phosphorylation of aquaporin-5 at its threonine 259 through cAMP signaling in salivary gland cells

Aquaporin-5 (AQP5), a water channel, plays key roles in salivary secretion. The novel phosphorylation of AQP5 was investigated by using human salivary gland (HSG) cells and mouse salivary glands. In the HSG cells stably transfected with a wild-type mouse AQP5 construct, a protein band immunoreactive with antibody against phosphorylated PKA substrate was detected in the AQP5 immunoprecipitated sample, and its intensity was enhanced by short-term treatment of the cells with 8-bromo-cAMP, forskolin, or phorbol 12-myristate 13-acetate, but not by that with A23187 calcium ionophore. Such enhancement was inhibited in the presence of H-89, a PKA inhibitor. An AQP5 mutant (AQP5-T259A) expressed by transfection of HSG cells was not recognized by anti-phosphorylated PKA substrate antibody, even when the cells were stimulated with the protein kinase activators. Immunoblotting and immunofluorescence studies using a specific antibody detecting AQP5 phosphorylated at its Thr259 demonstrated that AQP5 was rapidly and transiently phosphorylated at the apical membrane of acinar cells in the submandibular and parotid glands after administration of isoproterenol, but not pilocarpine. Furthermore, both AQP5 and AQP5-T259A were constitutively localized at the plasma membrane in HSG cells under the resting and forskolin-stimulated conditions. These results suggest that AQP5 is phosphorylated at its Thr259 by PKA through cAMP, but not Ca(2+), signaling pathways, and that this phosphorylation does not contribute to AQP5 trafficking in the salivary gland cells.     

The role of putative phosphorylation sites in the targeting and shuttling of the aquaporin-2 water channel

In renal collecting ducts, a vasopressin-induced cAMP increase results in the phosphorylation of aquaporin-2 (AQP2) water channels at Ser-256 and its redistribution from intracellular vesicles to the apical membrane. Hormones that activate protein kinase C (PKC) proteins counteract this process. To determine the role of the putative kinase sites in the trafficking and hormonal regulation of human AQP2, three putative casein kinase II (Ser-148, Ser-229, Thr-244), one PKC (Ser-231), and one protein kinase A (Ser-256) site were altered to mimic a constitutively non-phosphorylated/phosphorylated state and were expressed in Madin-Darby canine kidney cells. Except for Ser-256 mutants, seven correctly folded AQP2 kinase mutants trafficked as wild-type AQP2 to the apical membrane via forskolin-sensitive intracellular vesicles. With or without forskolin, AQP2-Ser-256A was localized in intracellular vesicles, whereas AQP2-S256D was localized in the apical membrane. Phorbol 12-myristate 13-acetate-induced PKC activation following forskolin treatment resulted in vesicular distribution of all AQP2 kinase mutants, while all were still phosphorylated at Ser-256. Our data indicate that in collecting duct cells, AQP2 trafficking to vasopressin-sensitive vesicles is phosphorylation-independent, that phosphorylation of Ser-256 is necessary and sufficient for expression of AQP2 in the apical membrane, and that PMA-induced PKC-mediated endocytosis of AQP2 is independent of the AQP2 phosphorylation state.     

Identification of a CK2 phosphorylation site in mdm2.

Mdm2 is a cellular oncoprotein the most obvious function of which is the down-regulation of the growth suppressor protein p53. It represents a highly phosphorylated protein but only little is yet known about the sites phosphorylated in vivo, the kinases that are responsible for the phosphorylation or the functional relevance of the phosphorylation status. Recently, we have shown that mdm2 is a good substrate for protein kinase CK2 at least in vitro. Computer analysis of the primary amino acid sequence of mdm2 revealed 19 putative CK2 phosphorylation sites. By using deletion mutants of mdm2 and a peptide library we identified the serine residue at position 269 which lies within a canonical CK2 consensus sequence (EGQELSDEDDE) as the most important CK2 phosphorylation site. Moreover, by using the mdm2 S269A mutant for in vitro phosphorylation assays this site was shown to be phosphorylated by CK2. Binding studies revealed that phosphorylation of mdm2 at S269 does not have any influence on the binding of p53 to mdm2.     

Protein kinase CK2 differentially phosphorylates maize chromosomal high mobility group B (HMGB) proteins modulating their stability and DNA interactions.

The high mobility group (HMG) proteins of the HMGB family are architectural factors in eukaryotic chromatin, which are involved in the regulation of various DNA-dependent processes. We have examined the post-translational modifications of five HMGB proteins from maize suspension cultured cells, revealing that HMGB1 and HMGB2/3, but not HMGB4 and HMGB5, are phosphorylated by protein kinase CK2. The phosphorylation sites have been mapped to the acidic C-terminal domains by analysis of tryptic peptides derived from HMGB1 and HMGB2/3 using nanospray ion trap mass spectrometry. In native HMGB1, Ser(149) is constitutively phosphorylated, whereas Ser(133) and Ser(136) are differentially phosphorylated. The functional significance of the CK2-mediated phosphorylation of HMGB proteins was analyzed by circular dichroism measurements showing that the phosphorylation increases the thermal stability of the HMGB proteins. Electrophoretic mobility shift assays demonstrate that the phosphorylation reduces the affinity of the HMGB proteins for linear DNA. The specific recognition of DNA minicircles is not affected by the phosphorylation, but a different pattern of protein-DNA complexes is formed. Collectively, these findings show that phosphorylation of residues within the acidic C-terminal domain of the HMGB proteins can modulate protein stability and the DNA binding properties of the HMGB proteins.     

The cytosolic protein kinase CK2 phosphorylates cardiac calsequestrin in intact cells

The luminal SR protein CSQ2 contains phosphate on roughly half of the serines found in its C-terminus. The sequence around phosphorylation sites in CSQ2 suggest that the in vivo kinase is protein kinase CK2, even though this enzyme is thought to be present only in the cytoplasm and nucleus. To test whether CSQ2 kinase is CK2, we combined approaches that reduced CK2 activity and CSQ2 phosphorylation in intact cells. Tetrabromocinnamic acid, a specific inhibitor of CK2, inhibited both the CSQ2 kinase and CK2 in parallel across a range of concentrations. In intact primary adult rat cardiomyocytes and COS cells, 24 h of drug treatment reduced phosphorylation of overexpressed CSQ2 by 75%. Down-regulation of CK2α subunits in COS cells using siRNA, produced a 90% decrease in CK2α protein levels, and CK2-silenced COS cells exhibited a twofold reduction in CSQ2 kinase activity. Phosphorylation of CSQ2 overexpressed in CK2-silenced cells was also reduced by a factor of two. These data suggested that CSQ2 in intact cells is phosphorylated by CK2, a cytosolic kinase. When phosphorylation site mutants were analyzed in COS cells, the characteristic rough endoplasmic reticulum form of the CSQ2 glycan (GlcNAc2Man9,8) underwent phosphorylation site dependent processing such that CSQ2-nonPP (Ser to Ala mutant) and CSQ2-mimPP (Ser to Glu mutant) produced apparent lower and greater levels of ER retention, respectively. Taken together, these data suggest CK2 can phosphorylate CSQ2 co-translationally at biosynthetic sites in rough ER, a process that may result in changes in its subsequent trafficking through the secretory pathway.     

Protein kinase CK2 phosphorylates the Fas-associated factor FAF1 in vivo and influences its transport into the nucleus

We previously identified the Fas-associated factor FAF1 as an in vitro substrate of protein kinase CK2 and determined Ser289 and Ser291 as phosphorylation sites. Here we demonstrate that these two serine residues are the only sites phosphorylated by CK2 in vitro, and that at least one site is phosphorylated in vivo. Furthermore, we analyzed putative physiological functions of FAF1 phosphorylation. The ability of FAF1 to potentiate Fas-induced apoptosis is not influenced by the FAF1 phosphorylation status; however, the nuclear import of a phosphorylation-deficient FAF1 mutant was delayed in comparison to wild-type FAF1.     

AMP-activated protein kinase phosphorylates Golgi-specific brefeldin A resistance factor 1 at Thr1337 to induce disassembly of Golgi apparatus

Sufficiency and depletion of nutrients regulate the cellular activities through the protein phosphorylation reaction; however, many protein substrates remain to be clarified. GBF1 (Golgi-specific brefeldin A resistance factor 1), a guanine nucleotide exchange factor for the ADP-ribosylation factor family associated with the Golgi apparatus, was isolated as a phosphoprotein from the glucose-depleted cells by using the phospho-Akt-substrate antibody, which recognizes the substrate proteins of several protein kinases. The phosphorylation of GBF1 was induced by 2-deoxyglucose (2-DG), which blocks glucose utilization and increases the intracellular AMP concentration, and by AICAR, an AMP-activated protein kinase (AMPK) activator. This phosphorylation was observed in the cells expressing the constitutively active AMPK. The 2-DG-induced phosphorylation of GBF1 was suppressed by Compound C, an AMPK inhibitor, and by the overexpression of the kinase-negative AMPK. Analysis using the deletion and point mutants identified Thr(1337) as the 2-DG-induced phosphorylation site in GBF1, which is phosphorylated by AMPK in vitro. ATP depletion is known to provoke the Golgi apparatus disassembly. Immunofluorescent microscopic analysis with the Golgi markers indicated that GBF1 associates with the fragmented Golgi apparatus in the cells treated with 2-DG and AICAR. The expression of the kinase-negative AMPK and the GBF1 mutant replacing Thr(1337) by Ala prevented the 2-DG-induced Golgi disassembly. These results indicate that GBF1 is a novel AMPK substrate and that the AMPK-mediated phosphorylation of GBF1 at Thr(1337) has a critical role, presumably by attenuating the function of GBF1, in the disassembly of the Golgi apparatus induced under stress conditions that lower the intracellular ATP concentration.     

Trafficking and phosphorylation dynamics of AQP4 in histamine-treated human gastric cells

BACKGROUND INFORMATION: AQP4 (aquaporin 4) internalization and a concomitant decrease in the osmotic water permeability coefficient (Pf) after histamine exposure has been reported in AQP4-transfected gastric HGT1 cells. RESULTS: In the present study we report that AQP4 internalization is followed by an increase in AQP4 phosphorylation. Histamine treatment for 30 min resulted in an approx. 10-fold increase in AQP4 phosphorylation that was inhibited by 1 microM H89, a specific PKA (protein kinase A) inhibitor, but not by PKC (protein kinase C) and CK2 inhibitors. Moreover, measurement of PKA activity after 30 min of histamine treatment showed that PKA activity was approx. 3-fold higher compared with basal conditions. AQP4 phosphorylation was prevented in cells treated with histamine for 30 min after pre-incubation with PAO (phenylarsine oxide), an inhibitor of protein endocytosis. Using an endo-exocytosis assay we showed that, after histamine washed out, internalized AQP4 recycled back to the cell surface, even in cells in which de novo protein synthesis was inhibited by cycloheximide. CONCLUSIONS: Phosphorylation experiments, combined with immunolocalization studies, indicated that AQP4 phosphorylation is mediated by PKA and occurs subsequently to its internalization in late endosomes. We suggest that phosphorylation might be a mechanism involved in retaining AQP4 in a vesicle-recycling compartment.     

Possible implication of the Golgi apparatus casein kinase in the phosphorylation of vesicle docking protein p115 Ser-940: a study with peptide substrates

Phosphorylation of human vescicle docking protein p115 at Ser-942 (homologous to Ser-940 in rat p115) promotes its dissociation from the Golgi membrane. Here we show that a peptide encompassing the 934--950 sequence of p115 is unaffected or poorly phosphorylated by a variety of Ser/Thr protein kinases with the notable exception of the Golgi apparatus casein kinase (G-CK) which phosphorylates it with an efficiency comparable to that of its optimal peptide substrates. In contrast phosphorylation of the p115 peptide by protein kinase CK2 is negligible compared to that of the specific peptide substrates of this kinase. Phosphorylation by G-CK is abolished if a conserved cluster of acidic residues at position between n + 4 and n + 9 (EDDDDE) is replaced by a neutral stretch (GAGAGA). These data strongly support the view that G-CK but not the other two classes of ubiquitous "casein kinases" (CK1 and CK2) is the natural phosphorylating agent of p115.     

Phosphorylation on the Ser 824 residue of TRPV4 prefers to bind with F-actin than with microtubules to expand the cell surface area

Previously, we demonstrated that the transient receptor potential vanilloid 4 (TRPV4) cation channel, a member of the TRP vanilloid subfamily, is one of the serum glucocorticoid-induced protein kinase1 (SGK1) authentic substrate proteins, and that the Ser 824 residue of TRPV4 is phosphorylated by SGK1 [1]. In this study, we demonstrated that phosphorylation on the Ser 824 residue of TRPV4 is required for its interaction with F-actin, using TRPV4 mutants (S824D; a phospho-mimicking TRPV4 mutant and S824A; a non-phosphorylatable TRPV4 mutant) and its proper subcellular localization. Additionally, we noted that the phosphorylation of the Ser824 residue promotes its single channel activity, Ca^2 + influx, protein stability, and cell surface area (expansion of plasma membrane).     

Polarized trafficking and surface expression of the AQP4 water channel are coordinated by serial and regulated interactions with different clathrin-adaptor complexes.

Aquaporin 4 (AQP4) is the predominant water channel in the brain. It is targeted to specific membrane domains of astrocytes and plays a crucial role in cerebral water balance in response to brain edema formation. AQP4 is also specifically expressed in the basolateral membranes of epithelial cells. However, the molecular mechanisms involved in its polarized targeting and membrane trafficking remain largely unknown. Here, we show that two independent C-terminal signals determine AQP4 basolateral membrane targeting in epithelial MDCK cells. One signal involves a tyrosine-based motif; the other is encoded by a di-leucine-like motif. We found that the tyrosine-based basolateral sorting signal also determines AQP4 clathrin-dependent endocytosis through direct interaction with the mu subunit of AP2 adaptor complex. Once endocytosed, a regulated switch in mu subunit interaction changes AP2 adaptor association to AP3. We found that the stress-induced kinase casein kinase (CK)II phosphorylates the Ser276 immediately preceding the tyrosine motif, increasing AQP4-mu 3A interaction and enhancing AQP4-lysosomal targeting and degradation. AQP4 phosphorylation by CKII may thus provide a mechanism that regulates AQP4 cell surface expression.     

Tyrosine-phosphorylated extracellular signal--regulated kinase associates with the Golgi complex during G2/M phase of the cell cycle: evidence for regulation of Golgi structure.

Phosphorylation of the extracellular signal-regulated kinases (ERKs) on tyrosine and threonine residues within the TEY tripeptide motif induces ERK activation and targeting of substrates. Although it is recognized that phosphorylation of both residues is required for ERK activation, it is not known if a single phosphorylation of either residue regulates physiological functions. In light of recent evidence indicating that ERK proteins regulate substrate function in the absence of ERK enzymatic activity, we have begun to examine functional roles for partially phosphorylated forms of ERK. Using phosphorylation site--specific ERK antibodies and immunofluorescence, we demonstrate that ERK phosphorylated on the tyrosine residue (pY ERK) within the TEY activation sequence is found constitutively in the nucleus, and localizes to the Golgi complex of cells that are in late G2 or early mitosis of the cell cycle. As cells progress through metaphase and anaphase, pY ERK localization to Golgi vesicles is most evident around the mitotic spindle poles. During telophase, pY ERK associates with newly formed Golgi vesicles but is not found on there after cytokinesis and entry into G1. Increased ERK phosphorylation causes punctate distribution of several Golgi proteins, indicating disruption of the Golgi structure. This observation is reversible by overexpression of a tyrosine phosphorylation--defective ERK mutant, but not by a kinase-inactive ERK2 mutant that is tyrosine phosphorylated. These data provide the first evidence that pY ERK and not ERK kinase activity regulates Golgi structure and may be involved in mitotic Golgi fragmentation and reformation.     

Phosphorylation and dephosphorylation of calsequestrin on CK2-sensitive sites in heart

Calsequestrin (CSQ) concentrates in junctional sarcoplasmic reticulum (SR) where it functions in regulation of Ca2+ release. When purified from heart tissue, cardiac CSQ contains phosphate on a cluster of C-terminal serine residues, but little is known about the cellular site of kinase action, and the identity of the kinase remains uncertain. To determine basic features of the phosphorylation, we examined the reaction in canine heart preparations. CSQ phosphorylation was observed in [32P]metabolically-labeled heart cells after adenoviral overexpression, and its constitutive phosphorylation was limited to a CK2-sensitive C-terminal serine cluster. The CSQ kinase was oriented intralumenally, as was CSQ, inside membrane vesicles, such that exposure to each required detergent permeabilization. Yet even after detergent permeabilization, CSQ was phosphorylated much less efficiently by protein kinase CK2 in cardiac microsomes than was purified CSQ. Reduced phosphorylation was strongly dependent upon protein concentration, and phosphorylation time courses revealed a phosphatase activity that occurred constitutively as phosphorylated substrate accumulates. Evidence of selective dephosphorylation of CSQ glycoforms in heart homogenates was also seen by mass spectrometry analysis. Molecules with greater mannose content, a feature of early secretory pathway compartments, were more highly phosphorylated, while greater dephosphorylation was apparent in more distal compartments. Taken together, the analyses of CSQ phosphorylation in heart suggest that a constitutive process of phosphate turnover occurs for cardiac CSQ perhaps associated with its intracellular transport.     

Cellular phosphorylation of an acidic proline-rich protein, PRP1, a secreted salivary phosphoprotein

Phosphorylation of many secreted salivary proteins is necessary for their biological functions. Identification of the kinase, which is responsible for in vivo phosphorylation, is complicated, because several of the protein phosphorylation sites conform both to the recognition sequence of casein kinase 2 (CK2) and Golgi kinase (G-CK), which both are found in the secretory pathway. This study was undertaken to determine the kinase recognition sequence in a secreted proline-rich salivary protein, PRP1, and thereby identify the responsible kinase. This was done by transfecting a human submandibular cell line, HSG, and a kidney cell line, HEK293, with expression vectors encoding wild-type or mutated PRP1. It was shown that phosphorylation occurred only at the same sites, Ser8 and 22, as in PRP1 purified from saliva. Phosphorylation at either site did not depend on the other site being phosphorylated. The sequence surrounding Ser8 has characteristics of both CK2 and G-CK recognition sequences, but destruction of the CK2 recognition site had no effect on phosphorylation, whereas no phosphorylation occurred if the G-CK recognition sequence was altered. The sequence surrounding Ser22 did not conform to any known kinase recognition sites. If Ser22 was mutated to Thr, no phosphorylation was seen, and a cluster of negatively charged residues at positions 27-29 was identified as part of the enzyme recognition site. Ser22 may be phosphorylated by a G-CK that recognizes an atypical substrate sequence or by a novel kinase. No difference in phosphorylation was seen between undifferentiated and differentiated HSG cells.     

Regulation of brain-type creatine kinase by AMP-activated protein kinase: Interaction,phosphorylation and ER localization

AMP-activated protein kinase (AMPK) and cytosolic brain-type creatine kinase (BCK) cooperate under energy stress to compensate for loss of adenosine triphosphate (ATP) by either stimulating ATP-generating and inhibiting ATP-consuming pathways, or by direct ATP regeneration from phosphocreatine, respectively. Here we report on AMPK-dependent phosphorylation of BCK from different species identified by in vitro screening for AMPK substrates in mouse brain. Mass spectrometry, protein sequencing, and site-directed mutagenesis identified Ser6 as a relevant residue with one site phosphorylated per BCK dimer. Yeast two-hybrid analysis revealed interaction of active AMPK specifically with non-phosphorylated BCK. Pharmacological activation of AMPK mimicking energy stress led to BCK phosphorylation in astrocytes and fibroblasts, as evidenced with a highly specific phospho-Ser6 antibody. BCK phosphorylation at Ser6 did not affect its enzymatic activity, but led to the appearance of the phosphorylated enzyme at the endoplasmic reticulum (ER), close to the ER calcium pump, a location known for muscle-type cytosolic creatine kinase (CK) to support Ca²⁺-pumping.     

Phosphorylation of conserved casein kinase sites regulates cAMP-response element-binding protein DNA binding in Drosophila

The Drosophila homolog of cAMP-response element-binding protein (CREB), dCREB2, exists with serine 231, equivalent to mammalian serine 133, in a predominantly phosphorylated state. Thus, unlike the mammalian protein, the primary regulation of dCREB2 may occur at a different step from serine 231 phosphorylation. Although bacterially expressed dCREB2 bound cAMP-response element sites, protein from Drosophila extracts was unable to do so unless treated with phosphatase. Phosphorylation of recombinant protein by casein kinase (CK) I or II, but not calcium-calmodulin kinase II or protein kinase A, inhibited DNA binding. Up to four conserved CK sites likely to be phosphorylated in vivo were responsible for this effect, and these sites were phosphorylated by a kinase present in Drosophila cell extracts that biochemically resembles CKII. We propose that the relative importance of different signaling pathways in regulating CREB activity may differ between Drosophila and mammals. In Drosophila, the dephosphorylation of CK sites appears to be the major regulatory step, while phosphorylation of serine 231 is necessary but secondary.     

Endothelin Induces a Calcium-Dependent Phosphorylation of PEA-15 in Intact Astrocytes: Identification of Ser104 and Ser116 Phosphorylated, Respectively, by Protein Kinase C and Calcium/Calmodulin Kinase II In Vitro

Abstract: PEA-15 (phosphoprotein enriched in astrocytes, M_r = 15,000) is an acidic serine-phosphorylated protein highly expressed in the CNS, where it can play a protective role against cytokine-induced apoptosis. PEA-15 is a major substrate for protein kinase C. Endothelins, which are known to exert pleiotropic effects on astrocytes, were used to analyze further the processes involved in PEA-15 phosphorylation. Endothelin-1 or endothelin-3 (0.1 µ M ) induced a robust phosphorylation of PEA-15 that was abolished by the removal of extracellular calcium, but only diminished by inhibitors of protein kinase C. Microsequencing of phosphopeptides generated by digestion of PEA-15 following endothelin-1 treatment identified two phosphorylated residues: Ser¹⁰⁴, previously recognized as the protein kinase C site, and a novel phosphoserine, Ser¹¹⁶, located in a consensus motif for either protein kinase casein kinase II or calcium/calmodulin-dependent protein kinase II (CaMKII). Partly purified PEA-15 was a substrate in vitro for CaMKII, but not for casein kinase II. Two-dimensional phosphopeptide mapping demonstrated that the site phosphorylated in vitro by CaMKII was also phosphorylated in intact astrocytes in response to endothelin. CaMKII phosphorylated selectively Ser¹¹⁶ and had no effect on Ser¹⁰⁴, but in vitro phosphorylation by CaMKII appeared to facilitate further phosphorylation by protein kinase C. Treatment of intact astrocytes with okadaic acid enhanced the phosphorylation of the CaMKII site. These results demonstrate that PEA-15 is phosphorylated in astrocytes by CaMKII (or a related kinase) and by protein kinase C in response to endothelin.