Nucleolar localization of phosphatidylinositol 4-kinase PI4K230 in various mammalian cells.

Background: Previous immunohistochemical investigations could not detect PI4K230, an isoform of mammalian phosphatidylinositol 4-kinases (also called type III alpha), in the nucleus and nucleolus of cells in spite of its predicted nuclear localization signals.

Methods: Immunofluorescent detection of PI4K230 and other PI4K isoforms was performed on formaldehyde (PFA) or ethanol fixed cells and rat brain cryosections. Costaining with nucleolin and the effect of siRNA, Triton X-100, DNase, and RNase treatments were also tested to determine the localization of PI4K230.

Subcellular localization and structural function of endogenous phosphorylated phosphatidylinositol 4-kinase (PI4K92).

Anti-phosphopeptide antibodies were raised against phosphatidylinositol 4-kinase (PI4K92) phosphorylation sites (Suer, S., Sickmann, A., Meyer, H.

Phosphorylation of human cardiac troponin I G203S and K206Q linked to familial hypertrophic cardiomyopathy affects actomyosin interaction in different ways.

cAMP-dependent protein kinase (PKA)-dependent phosphorylation of the two serine residues in the amino terminal region unique to cardiac troponin I (cTnI) is known to cause two effects: (i) decrease of the maximum Ca2+-controlled thin filament-activated myosin S1-ATPase (actoS1-ATPase) activity and mean sliding velocity of reconstituted thin filaments; (ii) rightward shift of the Ca2+ activation curves of actoS1-ATPase activity, filament sliding velocity, and force generation. We have studied the influence of phosphorylation of human wild-type cTnI and of two mutant cTnI (G203S and K206Q) causing familial hypertrophic cardiomyopathy (fHCM) on the secondary structure by circular dichroism spectroscopy and on the Ca2+ regulation of actin-myosin interaction using actoS1-ATPase activity and in vitro motility assays. Both mutations slightly influence the backbone structure of cTnI but only the secondary structure of cTnI-G203S is also affected by bis-phosphorylation of cTnI.

Mammalian phosphatidylinositol 4-kinases.

Three phosphatidylinositol 4-kinase isoforms, PI4K 230, 92 and 55 have been cloned and sequenced allowing a much wider characterization than the previously employed enzymological typing into type II and III enzymes. PI4K 230 and 92 contain a highly conserved catalytic core, PI4K55 one with a much lower degree of similarity. Candidate kinase motifs, deduced from the protein kinase super family, are absolutely conserved in all isoforms.

The ATP-binding site of brain phosphatidylinositol 4-kinase PI4K230 as revealed by 5'-p-fluorosulfonylbenzoyladenosine.

The ATP-binding site of purified bovine brain phosphatidylinositol 4-kinase 230 (PI4K230) was studied by its reaction with 5'-p-fluorosulfonylbenzoyladenosine (FSBA), an ATP-like alkylating reagent. Four hundred to eight hundred micromolar FSBA inactivated PI4K230 specifically with apparently first-order kinetics and resulted in 50% loss of enzyme activity in 36--130 min. The specificity of the reaction with FSBA was demonstrated by the lack of inactivation with 5'-p-fluorosulfonylbenzoyl chloride and by protection with ATP and ATP analogues against inactivation.

Human phosphatidylinositol 4-kinase isoform PI4K92. Expression of the recombinant enzyme and determination of multiple phosphorylation sites.

Human phosphatidylinositol 4-kinase, isoform PI4K92, was expressed as His6 tagged protein in Sf9 cells reaching a level of approximately 5% of cellular protein. The enzyme can be purified nearly to homogeneity in a single step by absorption/desorption on Ni/nitriloacetic acid agarose magnetic beads. High Km values in the millimolar range for ATP and PtdIns as well as only a moderate inhibition by adenosine and a sensitivity to Wortmannin (IC50 approximately 300 nM) characterize the enzyme as a type 3 PI4K.

Immunohistochemical localisation of two phosphatidylinositol 4-kinase isoforms, PI4K230 and PI4K92, in the central nervous system of rats.

The distribution and cellular localisation of the phosphatidylinositol 4-kinase isoforms, PI4K230 and PI4K92, that are believed to play important roles in the intracellular signalling mechanisms were studied in the rat brain (cortex, cerebellum, hippocampus and spinal cord) using immunocytochemistry with light and electron microscopy. PI4K230 was detected with a specific antibody purified by affinity chromatography from the egg yolk of chicken immunised with a 33-kDa fragment of bovine PI4K230, comprising amino acids 873-1175 of the native protein. PI4K92 was immunostained with a commercially available antibody raised in rabbit against amino acid residues 410-537 of human PI4K92.

Muscle phosphorylase kinase is not a substrate of AMP-activated protein kinase.

AMP-activated protein kinase (AMPK) and cAMP-dependent protein kinase (cAMPK) have been reported to phosphorylate sites on phosphorylase kinase (PhK). Their target residues Ser 1018 and Ser 1020, respectively, are located in the so-called multi-phosphorylation domain in the PhK alpha subunit. In PhK preparations, only one of these serines is phosphorylated, but never both of them.

Characterization of the cardiac holotroponin complex reconstituted from native cardiac troponin T and recombinant I and C.

Cardiac troponin I (cTnI), the inhibitory subunit of cardiac troponin (cTn), is phosphorylated by the cAMP-dependent protein kinase A at two adjacently located serine residues within the heart-specific N-terminal elongation. Four different phosphorylation states can be formed. To investigate each monophosphorylated form cTnI mutants, in which each of the two serine residues is replaced by an alanine, were generated.

Functional expression and characterisation of a new human phosphatidylinositol 4-kinase PI4K230.

By constructing DNA probes we have identified and cloned a human PtdIns 4-kinase, PI4K230, corresponding to a mRNA of 7.0 kb. The cDNA encodes a protein of 2044 amino acids.

Stepwise subunit interaction changes by mono- and bisphosphorylation of cardiac troponin I.

Four phosphorylation degrees of cardiac troponin I (cTnI) have been characterized, namely, a dephospho, a bisphospho, and two monophospho states. Here we describe for the first time a role of the monophosphorylated forms. We have investigated the interaction between the cardiac troponin subunits dependent on the phosphorylation state of cTnI by surface plasmon resonance (SPR) spectroscopy.

Phosphatidylinositol 4-kinases.

Polyphosphoinositides are involved in many signal transduction pathways in eukaryotic cells. The first committed step is catalysed by phosphatidylinositol 4-kinase leading to the formation of phosphatidylinositol 4-phosphate. In the last four years, ten cDNA molecules have been cloned which code isoforms of phosphatidylinositol 4-kinase; some of which are highly related.

Microanalysis and distribution of cardiac troponin I phospho species in heart areas.

Sequential phosphorylation and dephosphorylation of cTnI by the cAMP dependent protein kinase and by protein phosphatase 2A, respectively, produce the non-, mono- and bisphosphorylated species (Jaquet et al., 1995, Eur. J.

The identification of the phosphorylated 150/160-kDa proteins of sarcoplasmic reticulum, their kinase and their association with the ryanodine receptor.

In the present work we studied the relationship between the phosphorylated 150- and 160-kDa proteins and other SR proteins in the 150,000-170,000 range of molecular masses. on SDS-PAGE, the identification of their kinase, as well as the purification and structural interactions between these proteins and the rynodine receptor (RyR). The phosphorylated 150-kDa protein was identified as sarcalumenin based on: (a) its cross-reactivity with three different monoclonal antibodies specific for sarcalumenin.

VDAC/porin is present in sarcoplasmic reticulum from skeletal muscle.

In this study we demonstrate the existence of a protein with properties of the voltage-dependent anion channel (VDAC) in the sarcoplasmic reticulum (SR) using multiple approaches as summarized in the following: (a) 35 and 30 kDa proteins in different SR preparations, purified from other membranal systems by Ca2+/oxalate loading and sedimentation through 55% sucrose, cross-react with four different VDAC monoclonal antibodies. (b) Amino acid sequences of three peptides derived from the SR 35 kDa protein are identical to the sequences present in VDAC1 isoform. (c) Similar to the mitochondrial VDAC, the SR protein is specifically labeled by [14C]DCCD.

Bisphosphorylation of cardiac troponin I modulates the Ca(2+)-dependent binding of myosin subfragment S1 to reconstituted thin filaments.

We have reconstituted thin filaments comprising pyrene-labelled actin (pyr-actin), tropomyosin (Tm) and cardiac troponin (cTn). cTn was isolated in two defined phosphorylation states; completely dephosphorylated on all subunits and with only the cTnI subunit bisphosphorylated. The thin filament was saturated with cTn at a pyr-actin/Tm/cTn ratio of 7:1:1.

Identification of a 200 kDa polypeptide as type 3 phosphatidylinositol 4-kinase from bovine brain by partial protein and cDNA sequencing.

Two phosphatidylinositol 4-kinase isozymes, type 3 and type 2, have been separated on hydroxylapatite after solubilizing bovine brain microsomes with Triton X-114. Employing a newly developed renaturation procedure following SDS-PAGE, we demonstrate that a 200 kDa polypeptide carries the enzyme activity of this type 3 isoform. Chromatography on hydroxylapatite, Heparin-Sepharose, Superdex 200 and finally SDS-PAGE results in an approximately 30,000-fold purification.

Cloning of a phospholipase C-delta 1 of rabbit skeletal muscle.

The phospholipase C isoform responsible for the increase in the total myoplasmic inositol 1,4,5-trisphosphate concentration during tetanic contraction of isolated skeletal muscle and its mechanism of activation is not known. We have cloned and sequenced a phospholipase C cDNA of rabbit skeletal muscle coding for a protein of 745 amino acids with a molecular mass of 84,440 kDa. The deduced amino acid sequence exhibits the phospholipase C-specific domains X and Y which according to current knowledge very likely represent the catalytic centre of the enzyme.

Pattern formation on cardiac troponin I by consecutive phosphorylation and dephosphorylation.

Two serine residues located adjacently in the heart-specific N-terminus of cardiac troponin I can be phosphorylated in vivo. Both residues are sequentially phosphorylated and dephosphorylated by cAMP-dependent protein kinase (PKA) and protein phosphatase 2A (PP2A). The concentration changes of the different troponin I species have been determined separately for the phosphorylation and dephosphorylation reaction and approximated by time courses predicted by a reaction model.

Does phosphorylase kinase control glycogen biosynthesis in skeletal muscle?

Immunoblotting as well as enzyme assays demonstrate the presence of the self-glucosylating protein, glycogenin, in the protein-glycogen complex, in the sarcoplasmic reticulum and in phosphorylase kinase. In all three compartments glycogenin occurs in different, albeit, defined glucosylated forms, which upon deglucosylation are converted into a 42 kDa form. We suggest that phosphorylase kinase might have a dual function in glycogen biogenesis: firstly, control of glycogen degradation in the protein-glycogen complex via phosphorylation of glycogen phosphorylase b; secondly, regulation of glycogen biosynthesis on the sarcoplasmic reticular membranes via phosphorylation and thereby inhibition of glycogen synthase.

Endogenous, Ca(2+)-dependent cysteine-protease cleaves specifically the ryanodine receptor/Ca2+ release channel in skeletal muscle.

The association of an endogenous, Ca(2+)-dependent cysteine-protease with the junctional sarcoplasmic reticulum (SR) is demonstrated. The activity of this protease is strongly stimulated by dithiothreitol (DTT), cysteine and beta-mercaptoethanol, and is inhibited by iodoacetamide, mercuric chloride and leupeptin, but not by PMSF. The activity of this thiol-protease is dependent on Ca2+ with half-maximal activity obtained at 0.

Phosphoinositides in membranes that build up the triads of rabbit skeletal muscle.

The total membrane concentrations of PtdIns, PtdIns4P, and PtdIns(4,5)P2 contribute to the functional capacity of the Ins(1,4,5)P3 signalling system which is operating in skeletal muscle but the function of which is still unknown. Total amounts of these phosphoinositides have been determined in purified membranes of transverse tubules (TT) and terminal cisternae (TC) of the sarcoplasmic reticulum (SR) of rabbit skeletal muscle. PtdIns and PtdIns4P have been detected in both membrane systems whereas PtdIns(4,5)P2 (290 mumol/mol phospholipid) is confined only to TT.

Characterization of the cardiac troponin I phosphorylation domain by 31P nuclear magnetic resonance spectroscopy.

Cardiac holotroponin can be phosphorylated at serine 23 and/or 24 in the heart-specific region of bovine troponin I. When isolated freshly it is composed of a mixture of non-, two mono-, and bisphosphorylated species. At neutral pH the monophosphorylated form carrying phosphate at serine 24 yields a resonance signal at 4.

Interaction sites on phosphorylase kinase for calmodulin.

Holophosphorylase kinase was digested with Glu-C specific protease; from the peptide mixture calmodulin binding peptides were isolated by affinity chromatography and identified by N-terminal sequence analysis. Two peptides originating from the alpha subunit, having a high tendency to form a positively charged amphiphilic helix and containing tryptophane, were synthesized. Additionally, a homologous region of the beta subunit and a peptide from the alpha subunit present in a region deleted in the alpha' isoform were also selected for synthesis.

Identification of a new 80 k isoform of phosphatidylinositol 4-phosphate 5-kinase from bovine brain.

Phosphatidylinositol 4-phosphate 5-kinase is associated with bovine brain microsomes to an extent of approximately 65% of the total cellular enzyme activity. This membrane-associated kinase activity can be solubilized with Triton X-114. Following polyacrylamide gel electrophoresis in the presence of SDS the enzyme can be renaturated from gel slices in the presence of desoxycholate and Triton X-100.