Protein kinase casein kinase 2 holoenzyme produced ectopically in human cells can be exported to the external side of the cellular membrane

Ectokinases can phosphorylate extracellular proteins and external domains of membrane proteins influencing cell adhesion, movement, and cellular interactions. An ectokinase with the properties of casein kinase 2 (CK2) has been previously described, but little is known about the structural characteristics that allow this enzyme to be exported from the cell. Transfection of human embryonic kidney-293 cells with cDNAs coding for the catalytic (CK2alpha or CK2alpha') and regulatory (CK2beta) subunits with hemaglutinin tags allowed us to study the export of ectopically synthesized enzyme. When the catalytic (CK2alpha or CK2alpha') and the CK2beta regulatory subunits are cotransfected, the tetrameric enzyme composed of both subunits (holoenzyme) is detected outside the cell. This observation has been confirmed by assaying protein kinase activity in immunoprecipitates obtained with antihemaglutinin antibody by using a CK2-specific peptide substrate and by Western blots as well as by immunofluorescence of nonpermeabilized cells. Transfection with cDNA of catalytic or regulatory subunit alone does not result in export of these subunits. A study of the kinetics of appearance of the ectopically synthesized protein at different times after transfection indicates that a 5- to 7-h delay after the synthesis of the protein before it appears in the extracellular compartment. Using mutations of CK2alpha that eliminate phosphorylating activity [CK2alpha(Asp-156-Ala)] or that make it less sensitive to heparin inhibition [CK2alpha(Lys-75-Glu,Lys-76-Glu)] demonstrated that these mutations do not prevent the holoenzyme to be exported from the cells.     

Protein kinase A-anchoring (AKAP) domains in brefeldin A-inhibited guanine nucleotide-exchange protein 2 (BIG2)

Like other guanine nucleotide-exchange proteins (GEPs) that activate ADP-ribosylation factor (ARF) GTPases, brefeldin A-inhibited GEP2, BIG2, contains an approximately 200-aa Sec7 domain that is responsible for this catalytic activity and its inhibition by brefeldin A. The Sec7 domain is located near the center of the molecule and serves to accelerate replacement of GDP bound to ARF with GTP. To explore possible functions of the N-terminal region of BIG2 (1-832), we used three coding-region constructs as bait to screen a human heart cDNA library in a yeast two-hybrid system, retrieving two unique clones that encode a type I protein kinase A (PKA) regulatory subunit, RI alpha. Coimmunoprecipitation experiments confirmed interaction of in vitro translated BIG2 and RI alpha, as well as of the endogenous proteins in cytosol of cultured HepG2 cells. Using 28 deletion mutants, we found three regions of BIG2 that interacted with R subunits of PKA. Residues 27-48 (domain A) interacted with RI alpha and RI beta, 284-301 (domain B) interacted with RII alpha and RII beta, and 517-538 (domain C) interacted with RI alpha, RII alpha, and RII beta. Sequence analysis and helical wheel projection of amino acids in the three domains revealed potential amphipathic wheel structures characteristic for binding of PKA R subunits. Western blot analysis of subcellular fractions demonstrated translocation of BIG2 (and BIG1) from cytosol to the Golgi and other membrane structures after incubation of cells with 8-Br-cAMP or forskolin. All findings are consistent with a role for BIG2 as an A kinase-anchoring protein (or AKAP) that could coordinate cAMP and ARF regulatory pathways.     

Ca2+/calmodulin-dependent protein kinase II: identification of threonine-286 as the autophosphorylation site in the alpha subunit associated with the generation of Ca2+-independent activity.

Autophosphorylation of Ca2+/calmodulin-dependent protein kinase II converts the enzyme to a Ca2+-independent form. The time course for this conversion correlates with the autophosphorylation of a threonine residue located within a thermolytic phosphopeptide common to the alpha and beta/beta' subunits. In the present study, this site was identified in the alpha subunit. After autophosphorylation under conditions that produced near-maximal Ca2+-independent activity, the alpha and beta/beta' subunits were separated by NaDodSO4/PAGE, and the alpha subunit was cleaved with cyanogen bromide. The major phosphopeptide (CB-1), containing phosphothreonine as the only radiolabeled amino acid, was purified by reverse-phase high performance liquid chromatography and subjected to automated gas-phase Edman degradation. The sequence obtained, Xaa-Arg-Gln-Glu-Thr-Val-Asp-Xaa-Leu-Lys-Lys-Phe-Asn-Ala-Arg-Arg-Lys-Leu, represented the NH2-terminal 18 residues (residues 282-299) of a 26-amino acid cyanogen bromide peptide predicted from the deduced primary structure of the alpha subunit and contained a consensus sequence for Ca2+/calmodulin-dependent kinase II phosphorylation that included Thr-286. The sequences obtained for two phosphopeptides derived from secondary chymotryptic digestion of CB-1 confirmed that Thr-286 was the phosphorylated residue.     

Complete nucleotide sequence of dog heart creatine kinase mRNA: conservation of amino acid sequence within and among species.

Creatine kinase (CK; EC 2.7.3.2) plays an important role in energy metabolism in brain and muscle. Expression of CK isoenzymes is regulated during development and is tissue specific. To define the structures of canine CK isoenzymes and to elucidate the mechanism of regulation in their expression, CK cDNA clones from dog myocardium were isolated. Myocardial CK mRNA is predicted to encode a protein of 381 amino acids. The nontranslated regions of the mRNA comprise at least 38 bases at the 5' end and exactly 345 bases before the poly(A) tail. Partial protein sequences of dog muscle (M) CK and brain (B) CK subunits were determined and compared with the derived amino acid sequence of the myocardial enzyme and of M CK subunits of other species. The M CK subunits from different species share a very high degree (83-96%) of sequence identity. Dog M and B subunits share extensive sequence identity (74%), a degree of similarity not previously suspected. Southern blot analysis suggests that a CK gene family exists. These observations imply that evolutionary changes in the M CK subunit structure are constrained by the need for preservation of functional properties other than the kinase activity. This conservation is consistent with the possibility that the M subunit plays a structural role in cardiac and skeletal muscle.     

The structure of bovine F1-ATPase complexed with the peptide antibiotic efrapeptin.

In the previously determined structure of mitochondrial F1-ATPase determined with crystals grown in the presence of adenylyl-imidodiphosphate (AMP-PNP) and ADP, the three catalytic beta-subunits have different conformations and nucleotide occupancies. AMP-PNP and ADP are bound to subunits beta TP and beta DP, respectively, and the third beta-subunit (beta E) has no bound nucleotide. The efrapeptins are a closely related family of modified linear peptides containing 15 amino acids that inhibit both ATP synthesis and hydrolysis by binding to the F1 catalytic domain of F1F0-ATP synthase. In crystals of F1-ATPase grown in the presence of both nucleotides and inhibitor, efrapeptin is bound to a unique site in the central cavity of the enzyme. Its binding is associated with small structural changes in side chains of F1-ATPase around the binding pocket. Efrapeptin makes hydrophobic contacts with the alpha-helical structure in the gamma-subunit, which traverses the cavity, and with subunit beta E and the two adjacent alpha-subunits. Two intermolecular hydrogen bonds could also form. Intramolecular hydrogen bonds probably help to stabilize efrapeptin's two domains (residues 1-6 and 9-15, respectively), which are connected by a flexible region (beta Ala-7 and Gly-8). Efrapeptin appears to inhibit F1-ATPase by blocking the conversion of subunit beta E to a nucleotide binding conformation, as would be required by an enzyme mechanism involving cyclic interconversion of catalytic sites.     

The 90-kDa component of reticulocyte heme-regulated eIF-2 alpha (initiation factor 2 alpha-subunit) kinase is derived from the beta subunit of spectrin.

Antibodies from three different lines of monoclonal hybridomas crossreact with both the beta subunit of spectrin and the 90-kDa peptide present in highly purified preparations of the heme-controlled eIF-2 alpha (initiation factor 2 alpha-subunit) kinase from rabbit reticulocytes. Antibodies from two of the three lines enhance the enzymatic activity of the kinase preparation for phosphorylation of the alpha subunit of eukaryotic translational initiation factor 2 (eIF-2) and for phosphorylation of the 100-kDa peptide thought to be a peptide of the kinase that is phosphorylated during its activation. Also, it is shown that both the beta subunit of spectrin and the 90-kDa peptide can be phosphorylated by two protein kinases from reticulocytes, the catalytic subunit of cAMP-dependent protein kinase and a cAMP-independent protein kinase similar to casein kinase II. Furthermore, a phosphorylated 90-kDa peptide can be derived from phosphorylated beta subunit of spectrin by tryptic proteolysis. We conclude that the 90-kDa peptide is derived by proteolysis from the beta subunit of spectrin, probably from its carboxyl terminus, and suggest that the heme-sensitive eIF-2 alpha kinase, like the 56-kDa phosphatase [Wollny, E., Watkins, K., Kramer, G. & Hardesty, B. (1984) J. Biol. Chem. 259, 2484-2492], is associated with an element of the membrane skeleton in intact reticulocytes.     

Ca2+/calmodulin-dependent protein kinase II: identification of autophosphorylation sites responsible for generation of Ca2+/calmodulin-independence.

Ca2+/calmodulin-dependent protein kinase II contains two types of subunit, alpha (Mr 50,000) and beta (Mr 60,000/58,000), both of which undergo Ca2+/calmodulin-dependent autophosphorylation. Autophosphorylation is known to convert the enzyme to a Ca2+/calmodulin-independent form. In the present study, we have characterized the autophosphorylation sites on rat forebrain Ca2+/calmodulin-dependent protein kinase II that are most likely to be responsible for the generation of Ca2+/calmodulin-independence. Under conditions (0 degree C, low concentrations of ATP) sufficient to generate close to maximal Ca2+/calmodulin-independence, only a few of the phosphorylatable sites on the enzyme became phosphorylated. These autophosphorylation sites were examined by phospho amino acid analysis, two-dimensional thermolytic phosphopeptide mapping, and high-performance liquid chromatography. The time course of phosphorylation of threonine in both alpha and beta subunits was similar to the time course of the generation of Ca2+/calmodulin-independence. Moreover, the time course of phosphorylation of one set of peptides, referred to as peptide 1/1', present in both alpha and beta subunits was similar to the time course of the generation of Ca2+/calmodulin-independence. Threonine was the only amino acid phosphorylated in peptide 1/1'. An additional peptide, referred to as peptide 2, was phosphorylated in the beta subunit. The time course of phosphorylation of peptide 2, which also contained only phosphothreonine, did not parallel the time course of the generation of Ca2+/calmodulin-independence. It is likely that the phosphorylation of a threonine residue on peptide 1/1' is responsible for the generation of Ca2+/calmodulin-independence of Ca2+/calmodulin-dependent protein kinase II.     

Purification and NH2-terminal amino acid sequencing of the beta subunit of a human T-cell antigen receptor.

To obtain information about the structural basis for T-cell antigen recognition, a T3-associated Ti receptor molecule was isolated from crude membranes of the REX human thymic tumor line and purified by affinity chromatography with an anti- clonotypic monoclonal antibody in conjunction with preparative gel electrophoresis. NH2-terminal amino acid sequencing of the beta subunit unambiguously identified the amino acids in positions 2-12. Comparative protein sequence analysis by computer search demonstrated that this Ti beta sequence bore weak, but definite, homology to the first framework of the variable region of human lambda light chain. Anti-sera to a synthetic peptide corresponding to positions 2-11 precipitated the denatured Ti beta subunit from REX, thus confirming the above sequence. This information suggests that the Ti beta subunit is distantly related to human immunoglobulin lambda light chain and, moreover, should be of use in the molecular cloning of the Ti beta gene.     

Relationship between different enzymatic estimations of the infarct size

In 592 patients with an acute myocardial infarction different enzymatic estimations of the infarct size were compared. Heat stable lactate dehydrogenase (LD) was analysed every 12 hours for 48-108 hours. Aspartate amino transferase (ASAT) was analysed once daily for 3 days. In a subgroup creatine kinase (CK) and creatine kinase subunit B (CK B) were analysed every 6 hours for 48 hours. A strong positive correlation was observed between the peak activity of the different enzymes (r-values varying between 0.81 and 0.85). The peak activity of heat stable LD, CK and CK B were strongly related to the area under the enzyme curve (r-values varying between 0.94 and 0.98). A substantial decrease in estimated infarct size was observed in a large number of patients when intervals between sampling increased, most often found for CK B. It is concluded that the calculated peak activity of heat stable LD, ASAT, CK and CK B seems to give a similar information about the infarct size in the majority of patients with acute myocardial infarction. Analyses of the area under the enzyme curve do not deviate from calculated peak enzyme activity.     

Protein kinase CK2 modulates developmental functions of the abscisic acid responsive protein Rab17 from maize

The maize abscisic acid responsive protein Rab17 is a highly phosphorylated late embryogenesis abundant protein involved in plant responses to stress. In this study, we provide evidence of the importance of Rab17 phosphorylation by protein kinase CK2 in growth-related processes under stress conditions. We show the specific interaction of Rab17 with the CK2 regulatory subunits CK2 beta-1 and CK2 beta-3, and that these interactions do not depend on the phosphorylation state of Rab17. Live-cell fluorescence imaging of both CK2 and Rab17 indicates that the intracellular dynamics of Rab17 are regulated by CK2 phosphorylation. We found both CK2 beta subunits and Rab17 distributed over the cytoplasm and nucleus. By contrast, catalytic CK2 alpha subunits and a Rab17 mutant protein (mRab17) that is not a substrate for CK2 phosphorylation remain accumulated in the nucleoli. A dual-color image shows that the CK2 holoenzyme accumulates mainly in the nucleus. The importance of Rab17 phosphorylation in vivo was assessed in transgenic plants. The overexpression of Rab17, but not mRab17, arrests the process of seed germination under osmotic stress conditions. Thus, the role of Rab17 in growth processes is mediated through its phosphorylation by protein kinase CK2.     

CK MB versus total CK in the estimation of myocardial necrosis. Comparative kinetic analysis and evaluation of an immunological method]

It has been suggested that the adoption of a relatively specific marker of the myocardial cell, such as creatine kinase MB isoenzyme, can yield improved accuracy in estimating infarct size by serial serum sampling and compartmental analysis. Nevertheless, current methods for the evaluation of isoenzyme activity are cumbersome and unsuitable for clinical use. We have therefore employed a new test for the rapid determination of CK MB activity, based on the immunological inhibition of M subunities. In 19 patients not submitted either to intramuscular injection or to repeated defibrillations, a good correlation was found between indexes of necrosis based on MB and total CK determination (r = 0.94), with the cumulative MB release amounting to 16 +/- 4% of total CK. Significant differences were observed in 3 patients submitted to external cardiac massage (MB = 9 +/- 1% of total CK) thus suggesting a considerable extracardiac source of total CK due to the trauma of the skeletal muscle. The comparative kinetic analysis shows substantial differences between the two isoenzymes, not only concerning the greater disappearance rate of CK MB but, more significantly, related to a faster release of this isoenzyme from the myocardium, which has not been previously reported. The good correlations found between maximal appearance rate and cumulative enzyme release (r = 0.86) suggest that the former may represent an index of the rate of degradation of cellular membranes. Practical implications of these data are discussed.     

The modulatory action of loreclezole at the gamma-aminobutyric acid type A receptor is determined by a single amino acid in the beta 2 and beta 3 subunit.

Type A gamma-aminobutyric acid (GABAA) receptors of the mammalian nervous system are a family of ligand-gated ion channels probably formed from the coassembly of different subunits (alpha 1-6, beta 1-3, gamma 1-3, delta) in the arrangement alpha beta gamma or alpha beta delta. The activation of these receptors by GABA can be modulated by a range of compounds acting at distinct allosteric sites. One such compound is the broad-spectrum anticonvulsant loreclezole, which we have recently shown to act via a specific modulatory site on the beta subunit of the GABAA receptor. The action of loreclezole depends on the type of beta subunit present in the receptor complex; receptors containing beta 2 or beta 3 subunits have > 300-fold higher affinity for loreclezole than receptors containing a beta 1 subunit. We have used this property to identify the amino acid residue in the beta subunit that determines the subunit selectivity of loreclezole. Chimeric beta 1/beta 2 human GABAA receptor subunits were constructed and coexpressed in Xenopus oocytes with human alpha 1 and gamma 2s subunits. The chimera beta 1/beta 2Lys237-Gly334 conferred sensitivity to 1 microM loreclezole. Within this region there are four amino acids that are conserved in beta 2 and beta 3 but differ in beta 1. By mutating single amino acids of the beta 1 subunit to the beta 2/beta 3 equivalent, only the beta 1 mutation of Ser-290-->Asn conferred potentiation by loreclezole. Similarly, mutation of the homologous residue in the beta 2 and beta 3 subunits to the beta 1 equivalent (Asn-->Ser) resulted in loss of sensitivity to loreclezole. The affinity for GABA and the potentiation by flunitrazepam were unchanged in receptors containing the mutated beta subunits. Thus, a single amino acid, beta 2 Asn-289 (beta 3 Asn-290), located at the carboxyl-terminal end of the putative channel-lining domain TM2, confers sensitivity to the modulatory effects of loreclezole.     

Cloning, baculovirus expression, and characterization of a second mouse prolyl 4-hydroxylase alpha-subunit isoform: formation of an alpha 2 beta 2 tetramer with the protein disulfide-isomerase/beta subunit.

Prolyl 4-hydroxylase (EC 1.14.11.2) catalyzes the posttranslational formation of 4-hydroxyproline in collagens. The vertebrate enzyme is an alpha 2 beta 2 tetramer, the beta subunit of which is a highly unusual multifunctional polypeptide, being identical to protein disulfide-isomerase (EC 5.3.4.1). We report here the cloning of a second mouse alpha subunit isoform, termed the alpha (II) subunit. This polypeptide consists of 518 aa and a signal peptide of 19 aa. The processed polypeptide is one residue longer than the mouse alpha (I) subunit (the previously known type), the cloning of which is also reported here. The overall amino acid sequence identity between the mouse alpha (II) and alpha (I) subunits is 63%. The mRNA for the alpha (II) subunit was found to be expressed in a variety of mouse tissues. When the alpha (II) subunit was expressed together with the human protein disulfide-isomerase/beta subunit in insect cells by baculovirus vectors, an active prolyl 4-hydroxylase was formed, and this protein appeared to be an alpha (II) 2 beta 2 tetramer. The activity of this enzyme was very similar to that of the human alpha (I) 2 beta 2 tetramer, and most of its catalytic properties were also highly similar, but it differed distinctly from the latter in that it was inhibited by poly(L-proline) only at very high concentrations. This property may explain why the type II enzyme was not recognized earlier, as an early step in the standard purification procedure for prolyl 4-hydroxylase is affinity chromatography on a poly(L-proline) column.     

The first armadillo repeat is involved in the recognition and regulation of beta-catenin phosphorylation by protein kinase CK1

Multiple phosphorylation of beta-catenin by glycogen synthase kinase 3 (GSK3) in the Wnt pathway is primed by CK1 through phosphorylation of Ser-45, which lacks a typical CK1 canonical sequence. Synthetic peptides encompassing amino acids 38-64 of beta-catenin are phosphorylated by CK1 on Ser-45 with low affinity (K(m) approximately 1 mM), whereas intact beta-catenin is phosphorylated at Ser-45 with very high affinity (K(m) approximately 200 nM). Peptides extended to include a putative CK1 docking motif (FXXXF) at 70-74 positions or a F74AA mutation in full-length beta-catenin had no significant effect on CK1 phosphorylation efficiency. beta-Catenin C-terminal deletion mutants up to residue 181 maintained their high affinity, whereas removal of the 131-181 fragment, corresponding to the first armadillo repeat, was deleterious, resulting in a 50-fold increase in K(m) value. Implication of the first armadillo repeat in beta-catenin targeting by CK1 is supported in that the Y142E mutation, which mimics phosphorylation of Tyr-142 by tyrosine kinases and promotes dissociation of beta-catenin from alpha-catenin, further improves CK1 phosphorylation efficiency, lowering the K(m) value to <50 nM, approximating the physiological concentration of beta-catenin. In contrast, alpha-catenin, which interacts with the N-terminal region of beta-catenin, prevents Ser-45 phosphorylation of CK1 in a dose-dependent manner. Our data show that the integrity of the N-terminal region and the first armadillo repeat are necessary and sufficient for high-affinity phosphorylation by CK1 of Ser-45. They also suggest that beta-catenin association with alpha-catenin and beta-catenin phosphorylation by CK1 at Ser-45 are mutually exclusive.     

CK2 phosphorylation of eukaryotic translation initiation factor 5 potentiates cell cycle progression

Casein kinase 2 (CK2) is a ubiquitous eukaryotic Ser/Thr protein kinase that plays an important role in cell cycle progression. Although its function in this process remains unclear, it is known to be required for the G(1) and G(2)/M phase transitions in yeast. Here, we show that CK2 activity changes notably during cell cycle progression and is increased within 3 h of serum stimulation of quiescent cells. During the time period in which it exhibits high enzymatic activity, CK2 associates with and phosphorylates a key molecule for translation initiation, eukaryotic translation initiation factor (eIF) 5. Using MS, we show that Ser-389 and -390 of eIF5 are major sites of phosphorylation by CK2. This is confirmed using eIF5 mutants that lack CK2 sites; the phosphorylation levels of mutant eIF5 proteins are significantly reduced, relative to WT eIF5, both in vitro and in vivo. Expression of these mutants reveals that they have a dominant-negative effect on phosphorylation of endogenous eIF5, and that they perturb synchronous progression of cells through S to M phase, resulting in a significant reduction in growth rate. Furthermore, the formation of mature eIF5/eIF2/eIF3 complex is reduced in these cells, and, in fact, restricted diffusional motion of WT eIF5 was almost abolished in a GFP-tagged eIF5 mutant lacking CK2 phosphorylation sites, as measured by fluorescence correlation spectroscopy. These results suggest that CK2 may be involved in the regulation of cell cycle progression by associating with and phosphorylating a key molecule for translation initiation.     

Inhibition of CK2 activity by TGF-beta1 promotes IkappaB-alpha protein stabilization and apoptosis of immortalized hepatocytes

Nuclear factor kappaB (NF-kappaB) is an antiapoptotic factor involved in development, regeneration, and neoplastic progression of the liver. Previously, we have shown that stabilization of inhibitor kappaB (IkappaB)-alpha protein following treatment of hepatocytes with transforming growth factor (TGF)-beta1 promoted NF-kappaB repression, which then permitted induction of AP-1/SMAD-mediated liver cell death. Because basal IkappaB-alpha protein turnover is regulated by protein kinase CK2, here we have elucidated the regulation of CK2 kinase activity and its role in control of NF-kappaB levels following treatment with TGF-beta1. We show that both messenger RNA (mRNA) and protein levels of the CK2alpha catalytic subunit are down-regulated following TGF-beta1 stimulation in murine hepatocyte cells. The ensuing inhibition of CK2 kinase activity promotes stabilization of IkappaB protein, which is followed by the shutoff of constitutive NF-kappaB activity and induction of apoptosis. Ectopic expression of CK2alpha inhibits TGF-beta1-induced apoptosis through sustained activation of NF-kappaB. Conversely, expression of a kinase-dead mutant of CK2alpha potentiates TGF-beta1 cell killing. Importantly, we show that hepatocellular carcinomas (HCCs) derived from TGF-beta1 transgenic mice and human HCC cell lines display enhanced CK2 IkappaB kinase activity that contributes in part to an elevated NF-kappaB activity in vivo. In conclusion, inhibition of CK2 expression levels by TGF-beta1 is crucial for the induction of apoptosis of hepatocytes. Circumvention of this process by up-regulation of CK2 activity in transformed cells may contribute to the promotion of TGF-beta1-induced liver carcinogenesis.     

Activation of the fibrinogen binding site on platelets isolated from a patient with the Strasbourg I variant of Glanzmann's thrombasthenia

One proposed ligand binding site on platelet integrin alpha IIb beta 3 is the region of the beta 3 subunit encompassing amino acids 211-221. However, we recently showed that synthetic peptides corresponding to amino acids 211-221 inhibit fibrinogen binding to alpha IIb beta 3 by binding to alpha IIb beta 3 and not to fibrinogen. In this study, we show that AP6, a monoclonal antibody (MoAb) directed against amino acids 214-221 of beta 3, bound to immobilized active alpha IIb beta 3 but did not inhibit fibrinogen binding to the complex. We then determined whether nonfunctional alpha IIb beta 3 on platelets with a beta 3 Arg-214-->Trp mutation (Strasbourg I variant of Glanzmann's thrombasthenia or GTV) could be induced to aggregate after treatment with dithiothreitol (DTT). DTT has been shown to expose the fibrinogen receptor on normal platelets. DTT treatment of GTV platelets did result in the formation of the fibrinogen binding site as indicated by the binding of pI-55, an MoAb that only binds to the activated form of alpha IIb beta 3. Furthermore, DTT-treated GTV platelets aggregated in the presence of fibrinogen and divalent cations. This aggregation was inhibited by EDTA, RGDS, and the selective alpha IIb beta 3 antagonist, Ro 43-5054. These data show that Arg-214 of beta 3 is not required for fibrinogen binding or for platelet aggregation. However, this amino acid appears to be critical for the formation and for the maintenance of the correct tertiary structure of the fibrinogen binding site on alpha IIb beta 3.     

CK2 phosphorylates the angiotensin-converting enzyme and regulates its retention in the endothelial cell plasma membrane

Soluble angiotensin-converting enzyme (ACE) is derived from the membrane-bound form by proteolytic cleavage of its C-terminal domain. Because intracellular events might be involved in the regulation of the cleavage process, we determined whether the cytoplasmic tail of ACE is phosphorylated and whether this process regulates secretion. Immunoprecipitation of ACE (180 kDa) from (32)P-labeled endothelial cells revealed that ACE is phosphorylated. Phosphorylation was not observed in endothelial cells overexpressing a mutant form of ACE (ACEDeltaS, all five cytoplasmic serine residues replaced by alanine). CK2 coprecipitated with ACE from endothelial cells, and CK2 phosphorylated both ACE and a peptide corresponding to the cytoplasmic tail. Mutation of serine(1270) within the CK2 consensus sequence almost abolished ACE phosphorylation. In ACE-overexpressing endothelial cells, ACE was mostly localized to the plasma membrane. However, no ACE was detected in the plasma membrane of ACEDeltaS-overexpressing cells, although a precursor ACE (170 kDa) was prominent in the endoplasmic reticulum and the cell supernatant contained substantial amounts of the soluble protein (175 kDa). A correlation between ACE-phosphorylation and secretion was confirmed in endothelial cells treated with the CK2-inhibitor, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, which time-dependently decreased the phosphorylation of ACE and increased its shedding. These results indicate that the CK2-mediated phosphorylation of ACE regulates its retention in the plasma membrane and may determine plasma ACE levels.