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M Michalak

Publications and source records attributed to M Michalak.

At least 73 records · Page 4Linked to original sources
PubMed1995

Characterization of the recombinant C-terminal domain of dystrophin: phosphorylation by calmodulin-dependent protein kinase II and dephosphorylation by type 2B protein phosphatase.

We report that the C-terminal domain of skeletal muscle dystrophin expressed as a fusion protein with glutathione S-transferase (designated GST-CT-1) is a substrate for Ca2+/calmodulin-dependent phosphorylation and dephosphorylation. GST-CT-1 and GST-CT-1F (GST-CT-1 truncated by 20-25 residues) were phosphorylated by Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). The stoichiometries of phosphorylation by CaM kinase II were 1.65 mol of Pi/mol of GST-CT-1 and 0.39 mol of Pi/mol of GST-CT-1F, respectively, suggesting that the principal site(s) of phosphorylation is (are) located in the C-terminal 20-25 residues that are missing from GST-CT-1F. The GST-CT-1 fusion protein was phosphorylated on both serine and threonine residues, whereas GST-CT-1F was phosphorylated only on serine. CaM kinase II-phosphorylated GST-CT-1 and GST-CT-1F were efficiently dephosphorylated by calcineurin, a Ca2+/calmodulin-dependent protein phosphatase (type 2B protein phosphatase). Importantly, calcineurin was found to be associated with a purified sarcolemmal membrane preparation enriched in dystrophin. Type 2A protein phosphatase isolated from smooth muscle (SMP-I) and its catalytic subunit (SMP-ic) also dephosphorylated GST-CT-1, but were less active toward these substrates than was calcineurin. Type 2C phosphatase (SMP-II) and type 1 protein phosphatases [SMP-III, SMP-IV, and myosin-associated phosphatase (PP1M) of smooth muscle and skeletal muscle protein phosphatase 1c] were ineffective in dephosphorylating the C-terminal region of dystrophin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Calreticulin--the potential autoantigen in celiac disease.

Monoclonal antibodies to gliadin were recently found to cross-react with epitopes on rat enterocytes. Two molecules of mol. mass 62 and 66 kDa were isolated from enterocyte lysates by affinity chromatography using antigliadin monoclonal antibodies. The N-terminal amino acid sequence of the 62-kDa protein was determined to be XXXIYFKEQFLD. This amino acid sequence corresponds to amino acid sequence of rat calreticulin. The presence of calreticulin in enterocyte lysates was further confirmed using anticalreticulin serum. Anticalreticulin serum was also used to investigate the reactivity of isolated rat calreticulin. To analyze whether gliadin and calreticulin share similar epitopes recognized by anticalreticulin antibodies, synthetic dodecapeptides derived from the amino acid sequence of alpha gliadin were used in competitive ELISA assay. Two gliadin peptides, QEQVPLVQQQQF and YQLLQELCCQHL, were found to inhibit the binding of rabbit anti-rat calreticulin sera to rat calreticulin. The significant correlation was detected between IgA anticalreticulin and antigliadin antibodies (r = 0.827; P < 0.001) in celiac patients.

Amino Acid Sequence↗
PubMed1995

Calreticulin, an antithrombotic agent which binds to vitamin K-dependent coagulation factors, stimulates endothelial nitric oxide production, and limits thrombosis in canine coronary arteries.

Coagulation Factor IX/IXa has been shown to bind to cellular surfaces, and Factor IXa expresses its procoagulant activity by assembling into the intrinsic Factor X activating complex (Factors IXa/VIIIa/X), which also forms on membrane surfaces. This led us to identify cellular proteins which bind Factor IX/IXa; an approximately 55-kDa polypeptide was purified to homogeneity from bovine lung extracts based on its capacity to bind 125I-Factor IX in a dose-dependent and saturable manner. From protein sequence data of the amino terminus and internal peptides, the approximately 55-kDa polypeptide was identified as calreticulin, a previously identified intracellular calcium-binding protein. Recombinant calreticulin bound vitamin K-dependent coagulation factors, 125I-Factor IX, 125I-Factor X, and 125I-prothrombin (Kd values of approximately 2.7, 3.2, and 8.3 nM, respectively), via interaction with its C-domain, although it did not affect the coagulant properties of these proteins. 125I-Calreticulin also bound to endothelial cells in vitro (Kd approximately 7.4 nM), and mouse infusion studies showed an initial rapid phase of clearance in which calreticulin could be localized on the vascular endothelium. Exposure of endothelial cells to calreticulin led to dose-dependent, immediate, and sustained increase in the production of nitric oxide, as measured using a porphyrinic microsensor. In a canine electrically induced thrombosis model, intracoronary infusion of calreticulin (n = 7) prevented occlusion of the left circumflex coronary artery in a dose-dependent manner compared with vehicle-treated controls (n = 5). These results indicate that calreticulin interacts with the endothelium to stimulate release of nitric oxide and inhibit clot formation.

Animals↗
PubMed1995

Zn2+ binding to cardiac calsequestrin.

Zn2+ binding to canine cardiac calsequestrin was investigated using the Zn2+ specific fluorescence dye salicylcarbohydrazone (SACH), 65Zn2+ overlay and Zn(2+)-IDA chromatography. Cardiac calsequestrin binds approximately 200 moles of Zn2+/mole of protein with the Kd = 300 microM. Zn2+ binding to calsequestrin was further confirmed by 65Zn2+ overlay and Zn(2+)-dependent aggregation of the protein. However, calsequestrin did not bind to a Zn(2+)-IDA-agarose column, indicating that histidine residues may not be involved in Zn2+ binding to the protein. Circular dichroism revealed only minor Zn(2+)-dependent conformational changes in calsequestrin. We conclude that calsequestrin is a Ca(2+)- and Zn(2+)-binding protein and that Zn2+ may modulate the structure and function of the protein.

Animals↗
PubMed1995

Calreticulin modulates the in vitro DNA binding but not the in vivo transcriptional activation by peroxisome proliferator-activated receptor/retinoid X receptor heterodimers.

Calreticulin is a ubiquitous calcium binding/storage protein found primarily in the endoplasmic reticulum. Calreticulin has been shown to inhibit DNA binding and transcriptional activation by glucocorticoid and androgen hormone receptors by binding to the conserved sequence KXFF(K/R)R, present in the DNA-binding domains of all known members of the steroid/nuclear hormone receptor superfamily. To determine whether calreticulin might be a general regulator of hormone-responsive pathways, we examined its effect on DNA binding in vitro and transcriptional activation in vivo by heterodimers of the peroxisome proliferator-activated receptor (PPAR) and the 9-cis retinoic acid receptor (RXR alpha). We show here that purified calreticulin inhibits the binding of PPAR/RXR alpha heterodimers and of other nuclear hormone receptors, to peroxisome proliferator-responsive DNA elements in vitro. However, overexpression of calreticulin in transiently transfected cultured cells had little or no effect on transactivation mediated by PPAR/RXR alpha. Therefore, while calreticulin inhibits the binding of both nuclear and steroid hormone receptors to cognate response elements in vitro, our findings suggest that calreticulin does not necessarily play an important role in the regulation of all classes of hormone receptors in vivo.

Amino Acid Sequence↗
PubMed1994

Calreticulin: not just another calcium-binding protein.

In this paper we review some of the rapidly expanding information about calreticulin, a Ca(2+)-binding/storage protein of the endoplasmic reticulum. The emphasis is placed on the structure and function of calreticulin. We believe that calreticulin is a multifunctional Ca(2+)-binding protein and that distinct functional properties of the protein may be localized to each of the three structural domains of calreticulin. Most evidence indicates that calreticulin is a resident endoplasmic reticulum protein. However, it can also be found outside of the endoplasmic reticulum compartment, i.e. in the nuclear envelope, in the nucleus, in the cytotoxic granules in T-lymphocytes and in acrosomal vesicles of sperm cells. The evidence reviewed here clearly suggests that calreticulin has other functions in addition to its role as a Ca2+ storage protein in the endoplasmic reticulum.

Amino Acid Sequence↗
PubMed1994

Modulation of gene expression by calreticulin binding to the glucocorticoid receptor.

Calreticulin is a multifunctional protein that acts as a major Ca(2+)-binding (storage) protein in the lumen of the endoplasmic reticulum. It is also found in the nucleus, suggesting that it may have a role in transcription regulation. Calreticulin has been reported to bind to the synthetic peptide KLGFFKR, which is almost identical to an amino-acid sequence in the DNA-binding domain of the superfamily of nuclear receptors. Could calreticulin interact with the DNA-binding domain of these receptors and affect their function? Here we report that the amino terminus of calreticulin interacts with the DNA-binding domain of the glucocorticoid receptor and prevents the receptor from binding to its specific glucocorticoid response element. Overexpression of calreticulin in mouse L fibroblasts inhibits glucocorticoid-response-mediated transcriptional activation of a glucocorticoid-sensitive reporter gene and of the endogenous, glucocorticoid-sensitive gene encoding cytochrome P450. Together these results indicate that calreticulin may be important in gene transcription, regulating the glucocorticoid receptor and perhaps other members of the super-family of nuclear receptors.

Amino Acid Sequence↗
PubMed1994

2,4,6-Trinitrobenzenesulfonic acid modification of the carboxyl-terminal region (C-domain) of calreticulin.

The role of the primary amino groups of lysine sidechains in Ca2+ binding to calreticulin was evaluated by chemical modification of the amino group with 2,4,6-trinitrobenzenesulfonic acid (TNBS). TNBS binding to calreticulin could be described by two steps: (i) a fast reaction, with low affinity, and (ii) a slow reaction with a relatively high affinity. Inclusion of Ca2+ and/or Mg2+ decreased both the amount of TNBS bound to calreticulin and the apparent affinity constant of the slower reaction. In contrast, the properties of the faster reaction for TNBS binding were not sensitive to Ca2+ and/or Mg2+. Analysis of TNBS binding to the carboxyl-terminal (C-domain) and aminoterminal (N-domain) of calreticulin revealed that the C-domain and N-domain are responsible for the slow and fast component of the TNBS binding, respectively. In keeping with this, in the presence of Ca2+, TNBS binding to the C-domain was significantly reduced, whereas modification of the N-domain was unaffected. TNBS modification of calreticulin significantly decreased Ca2+ binding to the low affinity/high capacity Ca2+ binding site(s) which are localized to the C-domain but had no effect on the high affinity/low capacity Ca2+ binding localized to the N domain. In the C-domain of calreticulin, which contains the low affinity/high capacity Ca2+ binding sites, acidic residues are interspersed at regular intervals with one or more positively charged lysine and arginine residues. Our results indicate that the aminogroups of the lysine sidechains in the C-domain of calreticulin have a role in the low affinity/high capacity Ca2+ binding that is characteristic of this region of the protein and which is proposed to contribute significantly to the capacity of the endoplasmic reticulum Ca2+ store.

Amino Acid Sequence↗
PubMed1994

Crystallization of canine cardiac calsequestrin.

Calsequestrin is the major Ca2+ binding protein in the lumen of the sarcoplasmic reticulum membranes. Two X-ray quality crystal forms of canine cardiac calsequestrin were obtained by the hanging drop method using KCl as a precipitant. One form is monoclinic (space group P2(1), a = 73.4 A, b = 104.4 A, c = 60.2 A, beta = 120.4 degrees) with two molecules in the asymmetric unit and a solvent content of approximately 40%. The second form is trigonal (P3(1)21 or P3(2)21, a = b = 99.3 A, c = 89.8 A) with a single molecule in the asymmetric unit and 55% solvent content. Cross rotation function calculations show that despite the different space groups the packing of the molecules in both crystals is likely to be similar suggesting the existence of a stable dimer. The monoclinic crystals diffract beyond 3 A using a laboratory rotating anode source, while under the same conditions the trigonal crystals diffract only to approximately 4.5 A. This is the first report of successful preparation of X-ray quality crystals of a high capacity Ca2+ binding protein.

Animals↗
PubMed1994

Calreticulin: from Ca2+ binding to control of gene expression.

Calreticulin is a highly conserved Ca(2+)-binding/storage protein of the endoplasmic reticulum (ER). Recently, it has been shown to play a role in the control of gene expression by interacting with the DNA-binding domain of various steroid receptors. How does this ER protein gain access to the nuclear steroid receptors? We propose that calreticulin undergoes unique intracellular trafficking that allows it to colocalize with and bind to steroid receptors.

Journal Article↗
PubMed1994

Quantification of mitochondrial DNA in heteroplasmic fibroblasts with competitive PCR.

Kearns-Sayre syndrome (KSS) is a disease with severe clinical symptoms that often arises from a mitochondrial DNA deletion of 4977 bp. Quantification of defective mitochondrial DNA is important since the severity of symptoms in KSS is thought to be related to increased content of abnormal mitochondrial DNA. We developed a rapid, quantitative and competitive PCR assay to measure both wild-type and mutant forms of mitochondrial DNA in cells from KSS patients. The assay can accurately measure absolute numbers of mitochondrial DNA per cell by normalizing to a single copy nuclear gene.

Base Sequence↗
PubMed1994

Cell attachment to extracellular matrix substrates is inhibited upon downregulation of expression of calreticulin, an intracellular integrin alpha-subunit-binding protein.

We have demonstrated recently that calreticulin, an intracellular calcium-binding protein, can interact with the alpha-subunits of integrin receptors via the highly conserved KXGFFKR amino acid sequence present in the cytoplasmic domains of all integrin alpha-subunits (Rojiani et al. (1991) Biochemistry 30, 9859-9866). Here we demonstrate that calreticulin can be co-localized by immunofluorescence as well as co-purified with integrins, that recombinant calreticulin can also interact with integrins, and that the interaction occurs predominantly via the N-domain of calreticulin, to a much lesser extent with the C-domain, but not at all with the proline-rich P-domain. To demonstrate a physiological role for the interaction of calreticulin with integrins, calreticulin expression was downregulated by treating cells with antisense oligonucleotides designed to inhibit the initiation of translation of calreticulin. Antisense oligonucleotides, but not sense or non-sense oligonucleotides, inhibited attachment and spreading of cells cultured in the presence of fetal bovine serum, and also of cells plated on individual extracellular matrix substrates in the absence of serum. The antisense oligonucleotide inhibited cell proliferation of anchorage-dependent cells slightly, but there was no effect on cell viability. The effect on cell attachment was similar to that achieved by treating cells with an antisense oligonucleotide designed to inhibit translation of the integrin alpha 3 subunit, which resulted in the inhibition of cell attachment to alpha 3 beta 1-specific substrates. The effect of the antisense calreticulin oligonucleotide on cell attachment was demonstrated to be integrin-mediated since antisense calreticulin treatment of Jurkat cells abrogated the stimulation of collagen cell attachment achieved by attachment-stimulating signalling anti-alpha 2 (JBS2) and anti-beta 1 (21C8) antibodies. The oligonucleotides did not affect the rate of cell proliferation of these cells. These results demonstrate a fundamental role of calreticulin in cell-extracellular matrix interactions.

Amino Acid Sequence↗
PubMed1993

Phosphorylation of dystrophin. The carboxyl-terminal region of dystrophin is a substrate for in vitro phosphorylation by p34cdc2 protein kinase.

In this paper, we report that p34cdc2 protein kinase phosphorylates recombinant fragments of skeletal muscle dystrophin with a maximal incorporation of 1.8 mol of Pi/mol of protein. Phosphorylation of both serine and threonine residues occurs within the carboxyl-terminal 201 amino acids of dystrophin, with phosphothreonine localized to within 25 residues of the carboxyl terminus. Supporting these in vitro studies, we also show that native dystrophin is phosphorylated by p34cdc2 kinase in isolated sarcolemmal vesicles. Sequence analysis indicates two consensus sites for p34cdc2 protein kinase within the carboxyl-terminal 201 amino acids of dystrophin. Importantly, neither of these sites is conserved in dystrophin-related protein, and only one site is conserved in the 71-kDa alternative product of the Duchenne muscular dystrophy gene, despite an otherwise extremely high degree of sequence conservation between these proteins. Importantly, in this study we also show that dystrophin is phosphorylated in vivo in rat skeletal muscle primary cultures, and we suggest that further investigation of both in vivo and in vitro phosphorylation of this protein will comprise an important part in determination of its function(s).

Amino Acid Sequence↗
PubMed1993

Human liver calreticulin: characterization and Zn(2+)-dependent interaction with phenyl-sepharose.

A 60-kDa human calreticulin was isolated from liver homogenates. The protein was identified as calreticulin by its NH2-terminal amino acid sequence, by its mobility in SDS-PAGE, by its immunoreactivity with anti-calreticulin antibodies, by its Ca2+ binding, and by its localization to isolated ER membranes. In this study we show that Ca2+ binding to calreticulin results in Ca(2+)-dependent aggregation and precipitation of the protein. We also show that calreticulin and calsequestrin bind Zn2+ in 65Zn2+ overlay. In addition we have discovered that calreticulin exhibits a Zn(2+)-dependent interaction with hydrophobic matrix of phenyl-Sepharose that can be utilized in the purification of the protein.

Animals↗
PubMed1993

Interactions of calreticulin with proteins of the endoplasmic and sarcoplasmic reticulum membranes.

The ability of [125I]calreticulin to bind to membrane fractions isolated from different muscle and non-muscle tissues was examined by a protein overlay technique. Specific [125I]calreticulin binding proteins were detected in rat liver smooth and rough endoplasmic reticulum and Golgi, in canine pancreatic microsomes, and in rabbit skeletal muscle sarcoplasmic reticulum. These proteins were confined only to membranes that contain calreticulin; they were not found in rat liver mitochondria or cytosol. [125I]Calreticulin binds to a 50-kDa protein and a number of lower M(r) (20,000-38,000) endoplasmic reticulum membrane proteins and to 30-kDa protein in skeletal muscle sarcoplasmic reticulum. Full-length calreticulin and the carboxyl-terminal region (C-domain) of the protein both competed with [125I]calreticulin for binding to the membrane proteins. Binding of [125I]calreticulin to pancreatic microsomes was also partially inhibited by the N-domain and to a lesser extent by the P-domain of the protein. We conclude that calreticulin interacts with the endoplasmic reticulum membrane proteins mainly through its carboxyl-terminal domain and that the endoplasmic and sarcoplasmic reticulum membranes may contain different calreticulin binding proteins.

Animals↗
PubMed1992

Isolation and characterization of different C-terminal fragments of dystrophin expressed in Escherichia coli.

Dystrophin, the protein product of the Duchenne muscular dystrophy gene, is thought to belong to a family of membrane cytoskeletal proteins. Based on its deduced amino-acid sequence, it is postulated to have several distinct structural domains; an N-terminal region; a central, rod-shaped, domain; and a C-terminal domain [Koenig, Monaco & Kunkel (1988) Cell 53, 219-228]. The C-terminal domain is further divided into two regions; the first has some sequence similarity to slime mould alpha-actinin, and is rich in cysteine residues; this is followed by the C-terminal amino-acid sequence that is unique to dystrophin. Dystrophin is very difficult to purify in quantities sufficient for detailed studies of the structure/function relationships within the molecule. Therefore, in this study, we have expressed selected fragments of the C-terminal region of dystrophin, as fusion proteins, in Escherichia coli. Importantly, we describe the first successful purification, from E. coli lysates, of large quantities of fragments of dystrophin in a soluble form. The first fragment, termed CT-1, encodes the C-terminal 201 amino acids of the protein; the second, termed CT-2, spans the cysteine-rich region of the C-terminal domain. These fusion proteins were identified by their mobility in SDS/PAGE, by their interaction with appropriate affinity columns and by their reactivity with anti-dystrophin antibodies. The fragment CT-2, which spans a region containing putative EF-hand-like sequences, was found to bind Ca2+ in 45Ca2+ overlay experiments. In addition, we have discovered that the fragment CT-1, but not fragment CT-2, interacts specifically with the E. coli DnaK gene product [analogue of heat shock protein 70 (hsp70)]. This interaction is disrupted, in vitro, by the addition of ATP. Our results indicate that the two C-terminal fragments of dystrophin have differing biophysical properties, indicating that they may play distinct roles in the function of the protein.

Amino Acid Sequence↗
PubMed1992

Solubilization and purification of the prostaglandin E2 receptor from cardiac sarcolemma.

A prostaglandin E2 (PGE2) receptor was solubilized and isolated from cardiac sarcolemma membranes. Its binding characteristics are almost identical to those of the membrane bound receptor. [3H]PGE2 binding to solubilized and membrane bound receptor was sensitive to elevated temperature and no binding was observed in the absence of NaCl. No significant effects of DTT, ATP, Mg2+, Ca2+ or of changes in buffer pH were observed on [3H]PGE2 binding to either solubilized or membrane-bound receptor. Unlabelled PGE1 displaced over 90% of [3H]PGE2 from the CHAPS-solubilized receptor. PGD2, PGI2, PGF2 alpha and 6-keto-PGF1 alpha were not effective in displacing [3H]PGE2 from the receptor. Scatchard analysis of [3H]PGE2 binding to CHAPS-solubilized receptor revealed the presence of two types of PGE2 binding sites with Kd of 0.33 +/- 0.05 nM and 3.00 +/- 0.27 nM and Bmax of 0.5 +/- 0.04 and 2.0 +/- 0.1 pmol/mg of protein. The functional PGE2 receptor was isolated from CHAPS-solubilized SL membrane using two independent methods: first by a WGA-Sepharose chromatography and second by sucrose gradient density centrifugation. Receptor isolated by these two methods bound [3H]PGE2. Unlabelled PGE1 and PGE2 displaced [3H]PGE2 from the purified receptor. Scatchard analysis of [3H]PGE2 binding to purified receptor revealed the presence of the two binding sites as observed for the membrane bound and CHAPS-solubilized receptor. SDS-polyacrylamide gel electrophoresis of the purified receptor fractions revealed the presence of a protein band of M(r) of approx. 100,000. This 100-kDa was photolabelled with [3H]azido-PGE2, a photoactive derivative of PGE2. We propose that this 100-kDa protein is a cardiac PGE2 receptor.

Affinity Labels↗