Interaction of casein kinase II with ribosomal protein L22 of Drosophila melanogaster

The ubiquitous eukaryotic protein kinase CKII (casein kinase II) has been found to interact with a number of cellular proteins, either through the catalytic subunit or the regulatory subunit. Using the yeast two-hybrid screening method, we found that the catalytic subunit of Drosophila melanogaster CKII (DmCKII) interacts with Drosophila ribosomal protein L22 (rpL22). This interaction was also observed in vitro with a glutathione-S-transferase (GST)-rpL22 fusion protein. The predicted full-length Drosophila rpL22 protein has an N-terminal extension rich in alanine, lysine, and proline that appears to be unique to Drosophila. Deletion mapping revealed that the conserved core of rpL22 is responsible for the interaction with CKII. Moreover, purified DmCKII can phosphorylate a GST-L22 fusion protein at the C-terminal end, suggesting that this protein may be a substrate of CKII in Drosophila.     

The role of protein kinase CK2 in the regulation of the insulin production of pancreatic islets

An appropriate regulation of the insulin production and secretion in pancreatic β-cells is necessary for the control of blood glucose homeostasis. The pancreatic duodenal homeobox factor-1 (Pdx-1) is among the various factors and signals which are implicated in the regulation of the insulin synthesis and secretion in the pancreatic β-cells. Recently, we identified Pdx-1 as a substrate for protein kinase CK2. Since CK2 is implicated in the regulation of many different cellular signaling pathways we now asked whether it might also be involved in the regulation of the insulin regulation in β-cells. Here, we show that insulin treatment of β-cells resulted in an elevated CK2 kinase activity. On the other hand down-regulation of CK2 activity by quinalizarin led to an elevated level of insulin. These results demonstrate that CK2 is implicated in the insulin regulation on pancreatic β-cells.     

In vitro protein kinase activity measurement by flow cytometry

Protein kinases are important drug targets, and a wide variety of methods have been developed for assessing their activity. A key element in developing selective kinase inhibitors is the ability to rapidly compare the effects of an inhibitor on several related or unrelated kinases. We describe a simple, nonradioactive, bead-based method for detecting kinase activity in vitro. Biotinylated peptide substrates are immobilized on beads and phosphorylation is detected with anti-phosphopeptide antibodies with no separation steps required. Phosphorylation is dependent on the amount of kinase in the assay and can be inhibited by known kinase inhibitors in a concentration-dependent manner. Using Luminex technology, we measured the activity of three kinases (PKA, PKC-μ, and Akt) on multiple substrates simultaneously. We also discuss conditions necessary to optimize measurement of the activity of several kinases in a single sample.     

Molecular cloning and characterization of three cDNAs encoding putative mitogen-activated protein kinase kinases (MAPKKs) in Arabidopsis thaliana.

We isolated three Arabidopsis thaliana cDNA clones (ATMKK3, ATMKK4 and ATMKK5) encoding protein kinases with extensive homology to the mitogen-activated protein kinase kinases (MAPKKs) of various organisms in the catalytic domain. ATMKK3 shows high homology (85% identity) to NPK2, a tobacco MAPKK homologue. ATMKK4 and 5 are closely related to each other (84% identity). Phylogenetic analysis showed that the plant MAPKKs constitute at least three subgroups. The recombinant ATMKK3 and ATMKK4 were expressed as a fusion protein with glutathione S-transferase (GST) in Escherichia coli. Affinity purified GST-ATMKK3 and GST-ATMKK4 proteins contained phosphorylation activity, which shows that both the ATMKK3 and ATMKK4 genes encode functional protein kinases. Northern blot analysis revealed that the ATMKK3 gene expressed in all the organs. The levels of ATMKK4 and 5 mRNAs were relatively higher in steins and leaves than in flowers and roots. We determined the map positions of the ATMKK3, 4 and 5 genes on Arabidopsis chromosomes by RFLP mapping using P1 genomic clones.     

Degradation of protein kinase Cα and its free catalytic subunit, protein kinase M, in intact human neuroblastoma cells and under cell-free conditions: Evidence that PKM is degraded by mM calpain-mediated proteolysis at a faster rate than PKC

Proteolytic cleavage of protein kinase C (PKC) under cell-free conditions generates a co-factor independent, free catalytic subunit (PKM). However, the difficulty in visualizing PKM in intact cells has generated controversy regarding its physiological relevance. In the present study, treatment of SH-SY-5Y cells with 2-O-tetradecanoylphorbol 13-acetate resulted in complete down-regulation of PKC within 24 h without detection of PKM. By contrast, low levels of PKM were transiently detected following ionophore-mediated calcium influx under conditions which induced no detectable PKC loss. PKM was not detected during rapid cell-free degradation of partially purified SH-SY-5Y PKCα by purified human brain mM calpain. However, when the kinetics of PKC degradation were slowed by lowering levels of calpain, PKM was transiently detected. PKM was also only transiently observed following calpain-mediated degradation of purified rat brain PKCα. Densitometric analyses indicated that, once formed, PKM was degraded approximately 10 times faster than PKC. These data provide an explanation as to why PKM is difficult to observe in situ, and indicate that PKM should not be considered as an ‘unregulated’ kinase, since its persistence is apparently strictly regulated by proteolysis.     

Protein kinase CK2 phosphorylates the cell cycle regulatory protein Geminin

Geminin contributes to cell cycle regulation by a timely inhibition of Cdt1p, the loading factor required for the assembly of pre-replication complexes. Geminin is expressed during S and G2 phase of the HeLa cell cycle and phosphorylated soon after its synthesis. We show here that Geminin is an excellent substrate for protein kinase CK2 in vitro; and that the highly specific CK2 inhibitor tetrabromobenzotriazole (TBB) blocks the phosphorylation of Geminin in HeLa protein extracts and HeLa cells in vivo. The sites of CK2 phosphorylation are located in the carboxyterminal region of Geminin, which carries several consensus sequence motifs for CK2. We also show that a minor phosphorylating activity in protein extracts can be attributed to glycogen synthase kinase 3 (GSK3), which most likely targets a central peptide in Geminin. Treatment of HeLa cells with TBB does not interfere with the ability of Geminin to interact with the loading factor Cdt1.     

Phosphoenolpyruvate-dependent protein kinase activity in rat skeletal muscle

Phosphoenolpyruvate-dependent protein kinase activity has been demonstrated in the soluble fraction of rat skeletal muscle. The reaction was not due to the formation of ATP in the incubation mixture. Cyclic AMP, calcium, ATP and a number of phosphate acceptor proteins did not stimulate the reaction. One 32P-labelled protein (Mr 25000) was observed on SDS gels. The phosphorylated protein contained acid stable phosphoserine as a major phosphorylated amino acid. The phosphorylation reaction in crude extracts was not directly proportional to the amount of protein, but typical of a two-component system; i.e., kinase and substrate. The chromatography of soluble proteins on Ultrogel AcA44 separated the phosphate acceptor protein(s) from the phosphoenolpyruvate-dependent protein kinase activity.     

Mutation of the hydrophobic motif in a phosphorylation-deficient mutant renders protein kinase C delta more apoptotically active

Protein kinase C delta (PKCδ) is one of the important isoforms of PKCs that regulate various cellular processes, including cell survival and apoptosis. Studies have shown that activation of PKCδ is correlated with apoptosis in various cell types, depending upon various stimuli. Phosphorylation of Thr505, Ser643 and Ser662 is crucial in activation of PKCδ. Furthermore, phosphorylation of tyrosine residues, in particular that of Tyr311, is associated with PKCδ activation and induction of apoptosis. Here, we generated a hydrophobic motif phosphorylation-deficient mutant of PKCδ (PKCδ-S662A) by mutating Ser662 to Ala, and studied the effect of this mutation in inducing apoptosis in L929 murine fibroblasts. We report that this mutation renders PKCδ apoptotically more active. Furthermore, we found that the mutant PKCδ-S662A is tyrosine-phosphorylated and translocated to the membrane faster than its wild-type counterpart.     

Role of phospholipase D in the activation of protein kinase D by lysophosphatidic acid

Protein kinase D was auto-phosphorylated at Ser916 and trans-phosphorylated at Ser744/Ser748 in Rat-2 fibroblasts treated with lysophosphatidic acid. Both phosphorylations were inhibited by 1-butanol, which blocks phosphatidic acid formation by phospholipase D. The phosphorylations were also reduced in Rat-2 clones with decreased phospholipase D activity. Platelet-derived growth factor-induced protein kinase D phosphorylation showed a similar requirement for phospholipase D, but that induced by 4beta-phorbol 12 myristate 13-acetate did not. Propranolol an inhibitor of diacylglycerol formation from phosphatidic acid blocked the phosphorylation of protein kinase D, whereas dioctanoylglycerol induced it. The temporal pattern of auto-phosphorylation of protein kinase D closely resembled that of phospholipase D activation and preceded the trans-phosphorylation by protein kinase C. These results suggest that protein kinase D is activated by lysophosphatidic acid through sequential phosphorylation and that diacylglycerol produced by PLD via phosphatidic acid is required for the autophosphorylation that occurs prior to protein kinase C-mediated phosphorylation.     

Phosphorylation of Rho-associated kinase (Rho-kinase/ROCK/ROK) substrates by protein kinases A and C

Rho-associated kinase (Rho-kinase/ROCK/ROK) is a serine/threonine kinase and plays an important role in various cellular functions. The cAMP-dependent protein kinase (protein kinase A/PKA) and protein kinase C (PKC) are also serine/threonine kinases, and directly and/or indirectly take part in the signal transduction pathways of Rho-kinase. They have similar phosphorylation site motifs, RXXS/T and RXS/T. The purpose of this study was to identify whether sites phosphorylated by Rho-kinase could be targets for PKA and PKC and to find peptide substrates that are specific to Rho-kinase, i.e., with no phosphorylation by PKA and PKC. A total of 18 substrates for Rho-kinase were tested for phosphorylation by PKA and PKC. Twelve of these sites were easily phosphorylated. These results mean that Rho-kinase substrates can be good substrates for PKA and/or PKC. On the other hand, six Rho-kinase substrates showing no or very low phosphorylation efficiency (<20%) for PKA and PKC were identified. Kinetic parameters (K(m) and k(cat)) showed that two of these peptides could be useful as substrates specific to Rho-kinase phosphorylation.     

Physiological roles of mitogen-activated-protein-kinase-activated p38-regulated/activated protein kinase

Mitogen-activated protein kinases (MAPKs) are a family of proteins that constitute signaling pathways involved in processes that control gene expression, cell division, cell survival, apoptosis, metabolism, differentiation and motility. The MAPK pathways can be divided into conventional and atypical MAPK pathways. The first group converts a signal into a cellular response through a relay of three consecutive phosphorylation events exerted by MAPK kinase kinases, MAPK kinase, and MAPK. Atypical MAPK pathways are not organized into this three-tiered cascade. MAPK that belongs to both conventional and atypical MAPK pathways can phosphorylate both non-protein kinase substrates and other protein kinases. The latter are referred to as MAPK-activated protein kinases. This review focuses on one such MAPK-activated protein kinase, MAPK-activated protein kinase 5 (MK5) or p38-regulated/activated protein kinase (PRAK). This protein is highly conserved throughout the animal kingdom and seems to be the target of both conventional and atypical MAPK pathways. Recent findings on the regulation of the activity and subcellular localization, bona fide interaction partners and physiological roles of MK5/PRAK are discussed.     

Rat supernatant protein factor-like protein stimulates squalene monooxygenase and is activated by protein kinase A

Rat supernatant protein factor-like protein (SPF2) shares 90% sequence identity with rat SPF and 77% identity with human SPF, both of which have been shown to stimulate squalene monooxygenase in the cholesterol biosynthetic pathway. SPF2 appears to be predominantly expressed in respiratory and epithelial tissues, whereas SPF is expressed in liver. To determine if SPF2 was also able to stimulate squalene monooxygenase activity, we have cloned, expressed, and purified the protein following heterologous expression in Escherichia coli. SPF2 was only half as effective as SPF in stimulating squalene epoxidation and was more strongly inhibited by GTP and GDP. The inhibition by guanine nucleotides was fully prevented by alpha-tocopherol, a reported ligand for these proteins. Incubation of SPF2 with protein kinase A and ATP increased its activity by about twofold, has been found for SPF. These results indicate that SPF2 activity is modulated by guanine nucleotides and alpha-tocopherol, as well as by phosphorylation.     

A quantitative peptide array for evaluation of protein kinase activity

Peptide array, which is known as an emerging technology, has been developed for identification of protein kinase activity. For this purpose, the ability of quantitative analysis is very important because the absolute change in protein kinase activity is critical for the determination of cellular function. Here we report an original type of peptide array for quantitative evaluation of protein kinase activity by fluorescence imaging. We used the peptide array for the quantitative evaluation of the nonreceptor tyrosine kinase c-Src activity as a model for detecting protein kinase activities. By using positive and negative control peptides, we obtained the actual ratio of tyrosine phosphorylation of substrate peptide not only by purified c-Src but also by c-Src in cell lysate. In addition, the experimental approach provided simple immobilization of peptide. Our sensitive, specific, and high-throughput peptide array can be used for quantitative evaluation of kinase activity and potentially can be applied to drug discovery and screening.     

Human heart failure is accompanied by altered protein kinase A subunit expression and post-translational state

β-Adrenergic receptor blockade reduces total mortality and all-cause hospitalizations in patients with heart failure (HF). Nonetheless, β-blockade does not halt disease progression, suggesting that cAMP-dependent protein kinase (PKA) signaling downstream of β-adrenergic receptor activation may persist through unique post-translational states. In this study, human myocardial tissue was used to examine the state of PKA subunits. As expected, total myosin binding protein-C phosphorylation and Ser23/24 troponin I phosphorylation significantly decreased in HF. Examination of PKA subunits demonstrated no change in type II regulatory (RIIα) or catalytic (Cα) subunit expression, although site specific RIIα (Ser96) and Cα (Thr197) phosphorylation were increased in HF. Further, the expression of type I regulatory subunit (RI) was increased in HF. Isoelectric focusing of RIα demonstrated up to three variants, consistent with reports that Ser77 and Ser83 are in vivo phosphorylation sites. Western blots with site-specific monoclonal antibodies showed increased Ser83 phosphorylation in HF. 8-fluo-cAMP binding by wild type and phosphomimic Ser77 and Ser83 mutant RIα proteins demonstrated reduced Kd for the double mutant as compared to WT RIα. Therefore, failing myocardium displays altered expression and post-translational modification of PKA subunits that may impact downstream signaling.     

Structure of human protein kinase C eta (PKCeta) C2 domain and identification of phosphorylation sites

Protein kinase C eta (PKCeta) is one of several PKC isoforms found in humans. It is a novel PKC isoform in that it is activated by diacylglycerol and anionic phospholipids but not calcium. The crystal structure of the PKCeta-C2 domain, which is thought to mediate anionic phospholipid sensing in the protein, was determined at 1.75 A resolution. The structure is similar to that of the PKC epsilon C2 domain but with significant variations at the putative lipid-binding site. Two serine residues within PKC eta were identified in vitro as potential autophosphorylation sites. In the unphosphorylated structure both serines line the putative lipid-binding site and may therefore play a role in the lipid-regulation of the kinase.     

MOB control: reviewing a conserved family of kinase regulators

The family of Mps One binder (MOB) co-activator proteins is highly conserved from yeast to man. At least two different MOB proteins have been identified in every eukaryote analysed to date. Initially, yeast genetics revealed essential roles for Mob1p and Mob2p in the regulation of mitotic exit and cell morphogenesis. Studies in flies then showed that dMOB1/MATS is a core component of Hippo signalling. Loss of dMOB1 resulted in increased cell proliferation and decreased cell death, suggesting that MOB1 acts as tumour suppressor protein. Recent work focused primarily on mammalian cells has shown how hMOB1 can regulate NDR/LATS kinases, a function that can to be counteracted by hMOB2. Here we summarise and discuss our current knowledge of this emerging protein family, with emphasis on subcellular localisation, protein-protein interactions and biological functions in apoptosis, mitosis, morphogenesis, cell proliferation and centrosome duplication.     

Phosphorylation of troponin I by protein kinase C: Mechanism of inhibition by calmodulin and troponin C

The mechanism by which calmodulin and troponin C influence phosphorylation of troponin I (TnI) by protein kinase C was investigated. The phosphorylation of TnI by protein kinase C requires the presence of acidic phospholipid, calcium and diacylglycerol. Light scattering intensity and fluorescence intensity experiments showed that TnI associated with the phospholipid membranes and caused extensive aggregation. In the presence of Ca²⁺, TnI-phospholipid interactions were prevented by approximately stoichiometric amounts of either troponin C or calmodulin. Troponin C was shown to completely inhibit phosphorylation of TnI by either protein kianse C or by phosphorylase b kinase. In contrast, calmodulin completely inhibited phosphorylation of TnI by protein kinase C, but had only little effect on TnI phosphorylation by phosphorylase b kinase. Inhibition by calmodulin did not appear to be due to interaction with PKC, since calmodulin mildly increased protein kinase C phosphorylation of histone III-S. The ratio of phosphoserine to phosphothreonine in protein kinase C-phosphorylated TnI remained approximately constant for reactions inhibited by up to 90% by clamodulin. TnI interactions with phospholipid and phosphorylation of TnI by PKC were also prevented by high salt concentrations. However, salt concentrations adequate to inhibit phosphorylation were sufficient to dissociate only TnI, but not protein kinase C from the membrane. These results suggest that the binding of TnI to phospholipid is required for phosphorylation by protein kinase C and that prevention of this binding by any means completely inhibited phosphorylation of TnI by protein kinase C.