Autophosphorylation of type I phosphatidylinositol phosphate kinase regulates its lipid kinase activity

Phosphatidylinositol phosphate kinases (PIPKs) have important roles in the production of various phosphoinositides. For type I PIP5Ks (PIP5KI), a broad substrate specificity is known. They phosphorylate phosphatidylinositol 4-phosphate most effectively but also phosphorylate phosphatidylinositol (PI), phosphatidylinositol 3-phosphate, and phosphatidylinositol (3,4)-bisphosphate (PI(3, 4)P(2)), resulting in the production of phosphatidylinositol (4, 5)-bisphosphate (PI(4,5)P(2)), phosphatidylinositol 3-phosphate, phosphatidylinositol (3,4)-bisphosphate (PI(3,4)P(2)), phosphatidylinositol (3,5)-bisphosphate (PI(3,5)P(2)), and phosphatidylinositol (3,4,5)-trisphosphate. We show here that PIP5KIs have also protein kinase activities. When each isozyme of PIP5KI (PIP5KIalpha, -beta, and -gamma) was subjected to in vitro kinase assay, autophosphorylation occurred. The lipid kinase-negative mutant of PIP5KIalpha (K138A) lost the protein kinase activity, suggesting the same catalytic mechanism for the lipid and the protein kinase activities. PIP5KIbeta expressed in Escherichia coli also retains this protein kinase activity, thus confirming that no co-immunoprecipitated protein kinase is involved. In addition, the autophosphorylation of PIP5KI is markedly enhanced by the addition of PI. No other phosphoinositides such as phosphatidylinositol phosphate, phosphatidylinositol bisphosphate, or phosphatidylinositol trisphosphate have such an effect. We also found that the PI-dependent autophosphorylation strongly suppresses the lipid kinase activity of PIP5KI. The lipid kinase activity of PIP5KI was decreased to one-tenth upon PI-dependent autophosphorylation. All these results indicate that the lipid kinase activity of PIP5KI that acts predominantly for PI(4,5)P(2) synthesis is regulated by PI-dependent autophosphorylation in vivo.     

Autophosphorylation of cGMP-dependent protein kinase type II

Cyclic nucleotides are shown to stimulate the autophosphorylation of type II cGMP-dependent protein kinase (cGK) on multiple sites. Mass spectrometric based analyses, using a quadrupole time-of-flight-mass spectrometry instrument revealed that cGMP stimulated the in vitro phosphorylation of residues Ser110 and Ser114, and, at a slow rate, of Ser126 and Thr109 or Ser117, all located in the autoinhibitory region. In addition Ser445 was found to be phosphorylated in a cGMP-dependent manner, whereas Ser110 and Ser97 were already prephosphorylated to a large extent in Sf9 cells. cGMP-dependent phosphorylation of cGK II was also demonstrated in intact COS-1 cells and intestinal epithelium. Substitution of most of the potentially autophosphorylated residues for alanines largely abolished the cGMP stimulation of the autophosphorylation. Prolonged autophosphorylation of purified recombinant cGK II in vitro resulted in a 40-50% increase in basal kinase activity, but its maximal cGMP-stimulated activity and the EC50 for cGMP remained unaltered. Mutation of the major phosphorylatable serines 110, 114, and 445 into "phosphorylation-mimicking" glutamates had no effect on the kinetic parameters of cGK II. However, replacing the slowly autophosphorylated residue Ser126 by Glu rendered cGK II constitutively active. These results show that the fast phase of cyclic nucleotide-stimulated autophosphorylation of cGK II has a relatively small feed forward effect on its activity, whereas the secondary phase, presumably involving Ser126 phosphorylation, may generate a constitutively active form of the enzyme.     

Purification and properties of a protamine kinase and a type II casein kinase from bovine kidney mitochondria

Bovine kidney mitochondrial extracts contain an inactive protamine kinase and an inactive casein kinase. The protamine kinase was activated by chromatography on poly(L-lysine)-agarose. Two forms of this soluble mitochondrial protamine kinase were separated by chromatography on protamine-agarose. Both forms were purified about 80,000-fold to apparent homogeneity. Both forms of the protamine kinase consist of a single polypeptide chain with an apparent Mr approximately 45,000. Both enzyme forms underwent autophosphorylation without significant effect on activity, and both forms exhibited identical substrate specificities. The protamine kinase showed little activity toward branched-chain alpha-keto acid dehydrogenase (less than 3%), and it was essentially inactive (less than 0.1%) with pyruvate dehydrogenase, casein, and ovalbumin. The enzyme was active with histone H1 and with bovine serum albumin. Protamine kinase activity was unaffected by heparin (up to 100 micrograms/ml), by the protein inhibitor of cyclic AMP-dependent protein kinase, by Ca2+ and calmodulin, and by monoclonal antibody to the catalytic domain of protein kinase C from rat brain. The casein kinase was activated in the presence of spermine or by chromatography of the extract on DEAE-cellulose or poly(L-lysine)-agarose. The enzyme was purified about 80,000-fold to apparent homogeneity. It exhibited an apparent Mr 130,000 as determined by gel-permeation chromatography on Sephacryl S-300 in the presence of 0.5 M NaCl. Two subunits, with apparent Mr's 36,000 (alpha) and 28,000 (beta) were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The kinase underwent autophosphorylation of its beta-subunit, without significant effect on activity. Casein kinase activity was inhibited 50% by 1.5 micrograms/ml of heparin. Spermine (1.0 mM) stimulated activity of the purified kinase two- to three-fold at 1.5 mM Mg2+. Half-maximal stimulation occurred at 0.1 mM spermine. The kinase utilized both ATP and GTP as substrates. The casein kinase showed little activity (less than 1%) toward pyruvate dehydrogenase and branched-chain alpha-keto acid dehydrogenase from kidney mitochondria, and the kinase was essentially inactive with glycogen synthase a. The properties of this soluble mitochondrial kinase indicate that it is a type II casein kinase.     

Purification and characterization of bovine brain type I phosphatidylinositol kinase

Investigation into the phosphatidylinositol kinase activities in bovine brain has revealed the presence of a type I PtdIns kinase activity. This classification is based upon potent inhibition by neutral detergent and the production of a phosphatidylinositol phosphate that can be distinguished from phosphatidyl-inositol-4-phosphate [PtdIns(4)P] by thin-layer chromatography. The enzyme has been substantially purified and the activity is associated with an 85-kDa polypeptide on SDS/polyacrylamide gel electrophoresis. Analysis of the product confirms the identification of the enzyme as a type I PtdIns kinase. The purified kinase has been characterized with respect to substrate dependence (Mg2+, ATP, PtdIns), substrate presentation (pure lipid versus mixed micelle) and specificity [PtdIns versus PtdIns(4)P and phosphatidylinositol 4,5-bisphosphate].     

Tyrosine kinase signaling and type III effectors orchestrating Shigella invasion

Upon epithelial cell contact, Shigella type III effectors activate complex signaling pathways that induce localized membrane ruffling, resulting in Shigella invasion. Bacterial induced membrane ruffles require a timely coordination of cytoskeletal processes, including actin polymerization, filament reorganization and depolymerization, orchestrated by Rho GTPases and tyrosine kinases. An emerging concept is that multiple Shigella effectors act in synergy to promote actin polymerization in membrane extensions at the site of bacterial entry. Recent advances point to the role of Abl/Arg and Src tyrosine kinases as key regulators of bacterial induced cytoskeletal dynamics.     

Phosphorylation of tryptophanyl-tRNA-synthetase by casein kinase type II

Incubation of tryptophanyl-tRNA synthetase from bovine pancrease with [gamma-32P]ATP of [gamma-32P]GTP and casein kinase II from rabbit liver leads to the incorporation of labeled phosphate into serine residues of synthetase polypeptide. The maximal level of 32P incorporation into synthetase polypeptide (Mr = 60 kDa) 0.15 moles of 32P per 1 mole of polypeptide was observed. Electrophoretic analysis according to O'Farrell showed that kinase phosphorylates exclusively the most acidic polypeptides (pI 4.9) of the synthetase preparation. Pretreatment of synthetase with animal acidic and alkaline phosphatases had no influence on the level of 32P incorporation in synthetase during subsequent incubation in the presence of casein kinase II.     

Immunoaffinity purification of type I protein kinase C

We designed a simple procedure for the purification of type I protein kinase C, using immunoaffinity chromatography with a monoclonal antibody, MC-1b, obtained by rescreening hybridoma cells available for an affinity ligand. Western blotting demonstrated that MC-1b specifically reacted with type I protein kinase C, and the enzyme molecule dissociated from MC-1b-coupled Sepharose 4B with mild eluants such as thiocyanate retained the kinase activity. A 1148-fold purification was achieved and 210 micrograms of type I protein kinase C was obtained from three rabbit brains, by means of a two-step procedure, using DEAE-cellulose and immunoaffinity chromatography. The resultant preparation was homogeneous, as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis hydroxylapatite chromatography, and immunological analysis using MC-1a, MC-2a, and MC-3a.     

Sphingosine kinase type 2 activation by ERK-mediated phosphorylation

Sphingosine 1-phosphate (S1P), a potent lipid mediator, is a ligand for a family of five G protein-coupled receptors (S1P(1-5)) that have been shown to regulate a variety of biological responses important for cancer progression. The cellular level of S1P is low and tightly regulated in a spatio-temporal manner through its synthesis catalyzed by two sphingosine kinases, denoted SphK1 and SphK2. Many stimuli activate and translocate SphK1 to the plasma membrane by mechanisms that are dependent on its phosphorylation. Much less is known about activation of SphK2. Here we demonstrate that epidermal growth factor (EGF) as well as the protein kinase C activator, phorbol ester, induce rapid phosphorylation of hSphK2 which was markedly reduced by inhibition of MEK1/ERK pathway. Down-regulation of ERK1 blocked EGF-induced phosphorylation of SphK2. Recombinant ERK1 phosphorylated hSphK2 in vitro and increased its enzymatic activity. ERK1 also was found to be in a complex with hSphK2 in vivo. Site-directed mutagenesis indicated that hSphK2 is phosphorylated on Ser-351 and Thr-578 by ERK1 and that phosphorylation of these residues is important for EGF-stimulated migration of MDA-MB-453 cells. These studies provide the first clues to the mechanism of agonist-mediated SphK2 activation and enhance understanding of the regulation of SphK2 activity by phosphorylation and its role in movement of human breast cancer cells toward EGF.     

A fourth type of rabbit protein kinase C

Three rabbit cDNA clones coding for three types of protein kinase C (PKC alpha, beta, and gamma) have recently been identified and the structures determined [Ohno, S., Kawasaki, H., Imajoh, S., Suzuki, K., Inagaki, M., Yokokura, H., Sakoh, T., & Hidaka, H. (1987) Nature (London) 325, 161-166]. By use of these cloned cDNAs as hybridization probes, a fourth type (delta) of cDNA clone, which encodes a protein highly homologous to PKC alpha, beta, and gamma, was identified. PKC delta is composed of 697 amino acid residues and contains several peptide sequences determined at the protein level with the brain PKC preparation. This indicates that this molecular type (PKC delta) is, along with PKC alpha, beta, and gamma, a constituent of the brain PKC preparation. Sequence comparison among the four PKC types revealed that PKC delta is somewhat distinct from the other PKC types. PKC delta shows 99% amino acid sequence identity with rat PKC type I [Knopf, J. L., Lee, M.-H., Sultzman, L. A., Kriz, R. W., Loomis, C. R., Hewick, R. M., & Bell, R. M. (1986) Cell (Cambridge, Mass.) 46, 491-502], indicating relationship of these PKC types. The mRNA for PKC delta is exclusively concentrated in the brain.     

Identification of type I and type II inhibitors of c-Yes kinase using in silico and experimental techniques

c-Yes kinase is considered as one of the attractive targets for anti-cancer drug design. The DFG (Asp-Phe-Gly) motif present in most of the kinases will adopt active and inactive conformations, known as DFG-in and DFG-out and their inhibitors are classified into type I and type II, respectively. In the present study, two screening protocols were followed for identification of c-Yes kinase inhibitors. (i) Structure-based virtual screening (SBVS) and (ii) Structure-based (SB) and Pharmacophore-based (PB) tandem screening. In SBVS, the c-Yes kinase structure was obtained from homology modeling and seven ensembles with different active site scaffolds through molecular dynamics (MD) simulations. For SB-PB tandem screening, we modeled ligand bound active and inactive conformations. Physicochemical properties of inhibitors of Src kinase family and c-Yes kinase were used to prepare target focused libraries for screenings. Our screening procedure along with docking showed 520 probable hits in SBVS and tandem screening (120 and 400, respectively). Out of 5000 compounds identified from different computational methods, 2410 were examined using kinase inhibition assays. It includes 266 compounds (5.32%) identified from our method. We observed that 14 compounds (12%) are identified by the present method out of 168 that showed > 30% inhibition. Among them, three compounds are novel, unique, and showed good inhibition. Further, we have studied the binding of these compounds at the DFG-in and DFG-out conformations and reported the probable class (type I or type II). Hence, we suggest that these compounds could be novel drug leads for regulation of colorectal cancer.     

Purification of a yeast protein kinase sharing properties with type I and type II casein kinases

Cyclic nucleotide independent protein kinases preferring casein as in vitro substrates were resolved into four distinct species. Only one of the enzymes (CKII) was retained by DEAE-cellulose, whereas the three other enzymes (CKI-1, CKI-2, and CKI-3) were absorbed to CM-Sephadex, eluted with 250 and 600 mM NaCl, and fractionated by heparin-Sepharose chromatography. The casein kinase CKI-3 eluting at the highest NaCl concentration (550 mM) was purified to electrophoretic homogeneity by fast protein liquid chromatography. CKI-1 and CKI-2 correspond to mammalian type I casein kinase, because they bind to CM-Sephadex, they are monomeric enzymes of molecular weights below 50,000, they accept ATP exclusively (CKI-1) or predominantly (CKI-2) as phosphate donor, and they are either completely or relatively heparin insensitive. CKII corresponds to type II casein kinase due to its chromatographic properties, complex quaternary structure, nucleotide specificity (both ATP and GTP are phosphate donors), and heparin sensitivity. CKI-3 shares the following properties with type I casein kinases: it is retained by CM-Sephadex but not by DEAE-cellulose, and it consists of a monomeric protein having a molecular weight of 38,000. On the other hand, CKI-3 accepts both ATP and GTP with equal efficiency, and it is heparin sensitive (50% inhibition at 0.3 microgram/mL) like type II casein kinases. CKI-3 differs from the other three yeast casein kinases in requiring a low pH (5.5) and a high MgCl2 concentration (50 mM) for optimal activity. All four casein kinases phosphorylate their own catalytic protein at serine and threonine residues.     

Human liver type pyruvate kinase: cDNA cloning and chromosomal assignment

Pyruvate kinase (PK) has four isozymes (L,R,M1,M2) that are encoded mainly by two different genes. We isolated a cDNA clone from a Japanese adult liver lambda gt10 cDNA library by using a rat liver(L)-type PK cDNA probe. One positively hybridizing clone, hlPK-1, which contained a 1,049-base pair cDNA insert, was subjected to DNA sequence analysis. Comparisons of the sequence data with the rat PK cDNAs indicated that the cDNA encoded information for the carboxyl terminal 105 amino acids of a human L-type PK and a 3' untranslated region of 734 nucleotides. Furthermore, the karyotype analysis of several human-mouse hybrid cells and Southern blot analysis of DNAs of the hybrids with a hlPK-1 indicated that the human L-type PK gene is located on chromosome 1.     

Discovery of highly potent and selective type I B-Raf kinase inhibitors

A series of pyrazolo[1,5-α]pyrimidine analogs has been prepared and found to be potent and selective B-Raf inhibitors. Molecular modeling suggests they bind to the active conformation of the enzyme.     

Interaction between protein kinase Cmu and the vanilloid receptor type 1

The capsaicin receptor VR1 is a polymodal nociceptor activated by multiple stimuli. It has been reported that protein kinase C plays a role in the sensitization of VR1. Protein kinase D/PKCmu is a member of the protein kinase D serine/threonine kinase family that exhibits structural, enzymological, and regulatory features distinct from those of the PKCs, with which they are related. As part of our effort to optimize conditions for evaluating VR1 pharmacology, we found that treatment of Chinese hamster ovary (CHO) cells heterologously expressing rat VR1 (CHO/rVR1) with butyrate enhanced rVR1 expression and activity. The expression of PKCmu and PKCbeta1, but not of other PKC isoforms, was also enhanced by butyrate treatment, suggesting the possibility that these two isoforms might contribute to the enhanced activity of rVR1. In support of this hypothesis, we found the following. 1) Overexpression of PKCmu enhanced the response of rVR1 to capsaicin and low pH, and expression of a dominant negative variant of PKCmu reduced the response of rVR1. 2) Reduction of endogenous PKCmu using antisense oligonucleotides decreased the response of exogenous rVR1 expressed in CHO cells as well as of endogenous rVR1 in dorsal root ganglion neurons. 3) PKCmu localized to the plasma membrane when overexpressed in CHO/rVR1 cells. 4) PKCmu directly bound to rVR1 expressed in CHO cells as well as to endogenous rVR1 in dorsal root ganglia or to an N-terminal fragment of rVR1, indicating a direct interaction between PKCmu and rVR1. 5) PKCmu directly phosphorylated rVR1 or a longer N-terminal fragment (amino acids 1-118) of rVR1 but not a shorter one (amino acids 1-99). 6) Mutation of S116A in rVR1 blocked both the phosphorylation of rVR1 by PKCmu and the enhancement by PKCmu of the rVR1 response to capsaicin. We conclude that PKCmu functions as a direct modulator of rVR1.     

Phosphorylation of type II (beta) protein kinase C by casein kinase II.

Rat brain type II (beta) protein kinase C (PKC) was phosphorylated by rat lung casein kinase II (CK-II). Neither type I (gamma) nor type III (alpha) PKC was significantly phosphorylated by CK-II. CK-II incorporated 0.2-0.3 mol of phosphate into 1 mol of type II PKC. This phosphate was located at the single seryl residue (Ser-11) in the V1-variable region of the regulatory domain of the PKC molecule. A glutamic acid cluster was located at the carboxyl-terminal side of Ser-11, showing the consensus sequence for phosphorylation by CK-II. The velocity of this phosphorylation was enhanced by the addition of Ca2+, diolein, and phosphatidylserine, which are all required for the activation of PKC. Phosphorylation of casein or synthetic oligopeptides by CK-II was not affected by Ca2+, diolein, or phosphatidylserine. Available evidence suggests that CK-II phosphorylates preferentially the activated form of type II PKC. It remains unknown, however, whether this reaction has a physiological significance.     

Regulation of type IIalpha phosphatidylinositol phosphate kinase localisation by the protein kinase CK2.

Inositol lipid synthesis is regulated by several distinct families of enzymes [1]. Members of one of these families, the type II phosphatidylinositol phosphate kinases (PIP kinases), are 4-kinases and are thought to catalyse a minor route of synthesis of the multifunctional phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) from the inositide PI(5)P [2]. Here, we demonstrate the partial purification of a protein kinase that phosphorylates the type IIalpha PIP kinase at a single site unique to that isoform - Ser304. This kinase was identified as protein kinase CK2 (formerly casein kinase 2). Mutation of Ser304 to aspartate to mimic its phosphorylation had no effect on PIP kinase activity, but promoted both redistribution of the green fluorescent protein (GFP)-tagged enzyme in HeLa cells from the cytosol to the plasma membrane, and membrane ruffling. This effect was mimicked by mutation of Ser304 to alanine, although not to threonine, suggesting a mechanism involving the unmasking of a latent membrane localisation sequence in response to phosphorylation.     

Regulation of type II PIP kinase by PKD phosphorylation

The type II PIP kinases phosphorylate the poorly understood inositol lipid PtdIns5P, producing the multi-functional lipid product PtdIns(4,5)P(2). To investigate the regulation of these enzymes by phosphorylation, we partially purified a protein kinase from pig platelets that phosphorylated type IIalpha PIP kinase on an activation loop threonine residue, T376. Pharmacological studies suggested this protein kinase was protein kinase D (PKD), and in vitro experiments confirmed this identification. A phospho-specific antibody was developed and used to demonstrate phosphorylation of T376 in living cells, and its enhancement under conditions in which PKD was activated. Although we were unable to determine the effects of phosphorylation on PIP kinase activity directly, mutation of T376 to aspartate significantly inhibited enzyme activity. We conclude that the type II PIP kinases are physiological targets for PKD phosphorylation, and that this modification is likely to regulate inositol lipid turnover by inhibition of these lipid kinases.     

Autophosphorylation of rat liver type II cAMP-dependent protein kinase

A monoclonal antibody was prepared against the regulatory subunit (RII) of rat liver type II cAMP-dependent protein kinase. Autophosphorylated and nonphosphorylated RII in extracts from rat liver or hepatocytes were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and quantified by immunoblot analysis with this antibody. Under basal conditions, 90% of hepatocyte RII was in the phosphorylated form. Incubating hepatocytes with 8-bromo-cAMP and a phosphodiesterase inhibitor resulted in activation of cAMP-dependent protein kinase and glycogenolysis but did not affect phospho RII levels. RII phosphorylation was also unaffected by the inclusion of sufficient insulin to cause a decrease in cAMP-dependent protein kinase activity and glycogenolysis. The results indicate that unlike other cell types, dissociation of rat hepatocyte type II cAMP-dependent protein kinase does not result in dephosphorylation of RII. The biochemical basis for the apparent lack of RII dephosphorylation in intact hepatocytes was examined by comparison with smooth muscle where RII is rapidly dephosphorylated. Rat liver extract contained 4-fold less RII and had an 80-fold lower rate of dephosphorylation of endogenous RII compared to bovine smooth muscle extract. The differences in the rates of RII dephosphorylation in tissue extracts were not observed using purified RII from either tissue. These data suggested that the slow rate of RII dephosphorylation in rat hepatocytes is due to a difference in the susceptibility of endogenous rat liver RII to dephosphorylation rather than a difference in phosphatase activity.     

Endogenous dephosphorylation of synaptosomal calmodulin-dependent protein kinase type II

Calmodulin-dependent protein kinase Type II autophosphorylation in synaptosomes is localized to the cytoskeleton (synaptic junction), while a potent dephosphorylating activity is present in the lipid bilayer. The dephosphorylating activity is operative in intact synaptosomes and in a reconstitution system comprised of the cytoskeletal and Triton X-100 - soluble fractions. Dephosphorylation is inhibited by EDTA and pyrophosphate, but not by EGTA or NaF. The present characterization of endogenous synaptosomal dephosphorylating activity completes the regulatory cycle operating on this enzyme in which phosphorylation of calmodulin-dependent protein kinase type II inhibits its response to Ca+2 and calmodulin.