Colette Galand

Tyrosine phosphorylation regulates alpha II spectrin cleavage by calpain.

ABSTRACT Spectrins, components of the membrane skeleton, are implicated in various cellular functions. Understanding the diversity of these functions requires better characterization of the interacting domains of spectrins, such as the SH3 domain. Yeast two-hybrid screening of a kidney cDNA library revealed that the SH3 domain of αII-spectrin binds specifically isoform A of low-molecular-weight phosphotyrosine phosphatase (LMW-PTP). The αII-spectrin SH3 domain does not interact with LMW-PTP B or C nor does LMW-PTP A interact with the αI-spectrin SH3 domain. The interaction of spectrin with LMW-PTP A led us to look for a tyrosine-phosphorylated residue in αII-spectrin. Western blotting showed that αII-spectrin is tyrosine phosphorylated in vivo. Using mutagenesis on recombinant peptides, we identified the residue Y1176 located in the calpain cleavage site of αII-spectrin, near the SH3 domain, as an in vitro substrate for Src kinase and LMW-PTP A. This Y1176 residue is also an in vivo target for kinases and phosphatases in COS cells. Phosphorylation of this residue decreases spectrin sensitivity to calpain in vitro. Similarly, the presence of phosphatase inhibitors in cell culture is associated with the absence of spectrin cleavage products. This suggests that the Y1176 phosphorylation state could modulate spectrin cleavage by calpain and may play an important role during membrane skeleton remodeling.

The human red cell acid phosphatase is a phosphotyrosine protein phosphatase which dephosphorylates the membrane protein band 3

Human red cell cytosol acid phosphatase activity is supported by a main enzyme which can be extracted by DEAE and phosphocellulose chromatography. It uses pNPP as a substrate and is a protein phosphatase specific to phosphotyrosine. It dephosphorylates the tyrosine-phosphorylated cytosolic fragment of membrane protein 3. When taken together, these results suggest that the physiological role of red cell acid phosphatase is the FB3 phosphotyrosine dephosphorylation. Whatever it may be phosphotyrosine protein phosphatase activity is the first role of red cell acid phosphatase to be demonstrated.

Polymorphisms of the Tissue Factor Pathway Inhibitor (TFPI) Gene in Patients With Acute Coronary Syndromes and in Healthy Subjects Impact of the V264M Substitution on Plasma Levels of TFPI

-Mutations of the gene encoding tissue factor pathway inhibitor (TFPI), an inhibitor of TF-induced activation of the coagulation cascade, were screened for in 130 patients and 142 healthy controls to determine whether these variants contribute to acute coronary syndromes or modify plasma TFPI levels. The following 3 new polymorphisms were identified: 384T-->C in exon IV, which does not change the corresponding amino acid (tyrosine 57); -33C-->T in intron 7 (the T/T, C/T, and C/C genotypes were found in approximately 50%, 40%, and 10% of subjects in both groups); and 874G-->A in exon IX (GTG-->ATG), which predicts a valine to methionine change (V264M) in the carboxy-terminus tail of TFPI. The V264M polymorphism was found in 9.2% of the cases and 4.9% of the controls; the associated odds ratio (OR) for acute coronary syndromes was 2.0 (95% confidence interval [CI], 0.7 to 5.1). The OR increased to 3.6 (95% CI, 0.8 to 15.7) and 3.2 (95% CI, 0.9 to 11.8) in nonsmokers and patients without other risk factors, respectively. The possible link between the V264M polymorphism and coronary heart disease was checked in a large case-control study of myocardial infarction (Etude Cas-Témoins de lInfarctus du Myocarde [the ECTIM Study]). The results showed no link between the V264M polymorphism and coronary syndromes. Interestingly, however, 5 patients heterozygous for the V264M polymorphism had significantly lower plasma TFPI levels than did 13 patients with the most common genotype. Although our present results do not support an association between TFPI polymorphisms and acute coronary syndromes, the possibility that 1 of them, especially the exon IX polymorphism, is associated with subtypes of myocardial infarction or to evolutive particularities that were not assessed in this study, cannot be excluded and is currently being evaluated.

Heterogenous band 3 deficiency in hereditary spherocytosis related to different band 3 gene defects

Among 80 hereditary spherocytosis (HS) kindreds studied using denaturing electrophoretic separation of solubilized eythrocyte membrane proteins, we recognized three prominent subsets: HS with isolated spectrin deficiency, HS with combined spectrin and ankyrin deficiency, and HS with band 3 deficiency. These three subsets represent more than 80% of the HS kindreds studied. In this study, eight dominant HS kindreds with band 3 deficiency were investigated for band 3 mutations. In three of these kindreds, linkage analyses confirmed the band 3 gene as the culprit gene. In an attempt to identify the responsible mutations, denaturing gradient gel electrophoresis (DGGE) was used to explore the coding exons (exons 2–20) of band 3 gene. Five different mutations were found in the eight kindreds. In five kindreds we identified substitutions of highly conserved residues, positioned at boundaries of putative transmembrane segments: a Cu2003→u2003T substitution at codon 490 changed arginine (CGC) to cysteine (TGC) in three kindreds, a Cu2003→u2003T substitution at codon 837 changed threonine (ACG) to methionine (ATG) in two kindreds. In the sixth kindred a G deletion was found in a stretch of five G starting at position 1475, leading to a stop codon either at position 1527 or 1565. In the seventh kindred a T deletion at position 1600 resulted in a stop codon at position 1733 and in the last kindred a T deletion was identified at position 355, leading to a stop codon at position 447. The mutant transcript was present in HS patients bearing missense mutations, whereas only the normal transcript was found in HS patients with frameshift mutations. In the latter group the mean decrease in membrane band 3 content was significantly lower, leading to speculation that missense mutations may have some sort of dominant negative effect.

Molecular mechanism of erythrocyte pyruvate kinase deficiency

SummaryErythrocyte pyruvate kinase (PK) from 5 patients with congenital non-spherocytic hemolytic anemia and erythrocyte PK deficiency have been studied by immunological methods and electrofocusing.L type immunologically related PK was titrated in crude hemolysate with anti human liver L type PK rabbit serum and M2 type immunologically related PK with anti human leukocyte M2 type PK serum. After partial purification, molecular specific activity of erythrocyte PK was measured by immunoinactivation and electroimmunodiffusion with anti L type PK serum.Partially purified erythrocyte PK was focused on continuous sucrose gradient with 2% ampholines covering the pH range 5–8.PK enzymatic deficiency was due two times to a lowered molecular specific activity of the PK variants, the concentration of PK antigen being in the normal range.In the 3 other cases enzyme activity and immunological reactivity were likewise lowered. In the 2 patients with the most marked erythrocyte PK deficiency about 50% of the residual activity in crude hemolysate were non inhibited by anti L type PK serum, but were inhibited by anti M2 type PK serum.In 3 patients, the electrofocusing pattern of partially purified PK was significantly different from that of normal controls.In conclusion, the heterogeneity of the molecular mechanisms of the deficiency on the one hand, and the abnormalities of electrofocusing patterns on the other hand, seem to indicate that erythrocyte PK deficiency is due to the synthesis by muted structural genes of various abnormal PK molecules.The high ratio of M2 type PK in the most deficient hemolysates may be due to a compensatory process.

Purification and characterization of a casein kinase from human erythrocyte cytosol.

Abstract A casein kinase was extracted from human erythrocyte cytosol and purified by ammonium sulfate precipitation, chromatography on DEAE and phosphocellulose, and affinity chromatography on ATP-agarose. This enzyme did not use histone as a substrate; its activity was not stimulated by cyclic nucleotides. The pH of optimal activity was 6.5. The enzyme had an absolute requirement of Mg 2+ ions at an optimal concentration of 30 mM; activity was stimulated by Na + and K + at a maximal concentration of 0.125 M and inhibited by Ca 2+ . Casein was used as a substrate with a Km of 0.25 mg/ml; ATP was the preferential phosphoryl donor with a Km of 14.7 μM; GTP may be used with a lower yield and a Km of 26.3 μM. ADP was a competitive inhibitor of ATP with a Ki of 14 μM. 2–3 DPG was an allosteric inhibitor of ATP with an apparent Ki of 4.6 mM and a Hill coefficient of 3.8. Kinetic data indicate that the reaction follows a coordinated mechanism with ATP as the first substrate and subsequent formation of a ternary complex with the protein. SDS-PAGE of the purified enzyme showed two different peptide chains of molecular weight 35 000 and 25 000.

In vitro digestion of spectrin, protein 4.1 and ankyrin by erythrocyte calcium dependent neutral protease (calpain I)

1. In whole ghosts, ankyrin, protein 4.1, protein band 3 and spectrin are lysed by purified calpain I in the presence of calcium. 2. Limited calpain lysis of purified ankyrin results in several peptides, including a 85 kD peptide bearing the ankyrin interaction site for the protein band 3 internal fragment (43 kD), and a 55 kD peptide carrying the ankyrin-spectrin interaction site. 3. These peptides are differently phosphorylated: the 85 kD by cytosol casein kinase, and the 55 kD by membrane casein kinase. 4. Protein 4.1 lysis mainly produces a 30 kD peptide resistant to proteolysis. 5. The spectrin beta-chain is more sensitive to calpain cleavage than the alpha chain; both chains seem to be cleaved in a similar sequential manner. 6. Limited proteolysis of spectrin dimer does not impede tetramerization in vitro.

In vitro phosphorylation of the red blood cell cytoskeleton complex by cyclic AMP-dependent protein kinase from erythrocyte membrane.

Hydrosoluble proteins extracted from human erythrocyte ghosts by dialysis at low ionic strength and alkaline pH contain a cyclic AMP-dependent protein kinase which phosphorylates in vitro the cytoskeleton components in crude extracts. Spectrin components 1 and 2, actin and protein band 4.1 undergo this cyclic AMP-dependent endogenous phosphorylation together with low molecular weight peptides solubilized with the cytoskeleton in hydrosoluble extract. However, pure spectrin and purified erythrocyte G-actin were not phosphorylated by purified cyclic AMP-dependent protein kinase from erythrocyte membrane. Purified G-actin when added free to crude extract does not undergo phosphorylation by the cyclic AMP-dependent protein kinase present in this extract. In contrast, purified cyclic AMP-dependent protein kinase added either to crude extract or to the purified cytoskeleton complex (spectrin, actin and protein band 4.1), phosphorylates spectrin, actin and protein band 4.1. We can conclude that (1) cyclic AMP-dependent phosphorylation of red cell cytoskeleton occurs in vitro only when the cytoskeleton components are in a complexed form; (2) red cell actin, like other cellular actins, may be phosphorylated by cyclic AMP-dependent protein kinase but only in the oligomeric form and not in the G form.

Molecular basis of clinical and morphological heterogeneity in hereditary elliptocytosis (HE) with spectrin αI variants

Summary. The impaired ability of spectrin dimers to self‐associate into tetramers is one of the most frequent defects associated with hereditary elliptocytosis (HE) and its more serious form, hereditary pyropoikylocytosis (HPP). We previously described four proteic variants of the spectrin (Sp) a1 tryptic domain associated with the Sp dimer self‐association defect (Sp α1/78, Sp α1/74, Sp α1/65, Sp α1/46 variants). Following the characterization of proteic variants, genomic molecular defects were identified and most of the mutations appeared to lie either in or near the self‐association site, i.e. in the αI tryptic domain or in the βI tryptic domain.

Purification and characterization of an adenosine cyclic 3′:5′ monophosphate-dependent protein kinase from human erythrocyte membrane

Abstract A cAMP dependent protein kinase was extracted from human erythrocyte membrane with hydrosoluble fraction and partially purified by ammonium sulfate-precipitation and DEAE-cellulose chromatography. The pH of optimal activity is 6.5; the enzyme has an absolute requirement of Mg 2+ ions at the concentration of 10 mM and is strongly inhibited by Ca 2+ . It uses ATP as phosphate donor with a Km of 3.7 × 10 −6 M. Cyclic AMP stimulates the activity with an apparent Ka of 5 × 10 −8 M; cIMP and cGMP also acts as activators. Enzyme activity is thermolabile and not protected by Mg ATP complex. The enzyme purified from erythrocyte membrane is a type I protein-kinase as proven by DEAE cellulose chromatography and dissociation of the subunits in presence of NaCl 0.5 M and histone.