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Biomedical subjects

C M Cohen

Publications and source records attributed to C M Cohen.

At least 19 recordsLinked to original sources
PubMed1999

Mild spherocytosis and altered red cell ion transport in protein 4. 2-null mice.

Protein 4.2 is a major component of the red blood cell (RBC) membrane skeleton. We used targeted mutagenesis in embryonic stem (ES) cells to elucidate protein 4.2 functions in vivo. Protein 4. 2-null (4.2(-/-)) mice have mild hereditary spherocytosis (HS). Scanning electron microscopy and ektacytometry confirm loss of membrane surface in 4.2(-/-) RBCs. The membrane skeleton architecture is intact, and the spectrin and ankyrin content of 4. 2(-/-) RBCs are normal. Band 3 and band 3-mediated anion transport are decreased. Protein 4.2(-/-) RBCs show altered cation content (increased K+/decreased Na+)resulting in dehydration. The passive Na+ permeability and the activities of the Na-K-2Cl and K-Cl cotransporters, the Na/H exchanger, and the Gardos channel in 4. 2(-/-) RBCs are significantly increased. Protein 4.2(-/-) RBCs demonstrate an abnormal regulation of cation transport by cell volume. Cell shrinkage induces a greater activation of Na/H exchange and Na-K-2Cl cotransport in 4.2(-/-) RBCs compared with controls. The increased passive Na+ permeability of 4.2(-/-) RBCs is also dependent on cell shrinkage. We conclude that protein 4.2 is important in the maintenance of normal surface area in RBCs and for normal RBC cation transport.

Animals↗
PubMed1998

Guanine nucleotide-dependent translocation of RhoA from cytosol to high affinity membrane binding sites in human erythrocytes.

The translocation of the small GTP-binding protein Rho from the cytosolic to membrane-bound form is an early step in many cellular signal-transduction events, but little is known regarding the mechanism of Rho association with the plasma membrane. We have used membranes from human erythrocytes to uncover a novel class of integral membrane components involved in the Rho-membrane association. Membranes of human erythrocytes contain several proteins of the Ras superfamily. Using specific antibodies and C3 exoenzyme of Clostridium botulinum we have identified one of them as RhoA. This protein was detected in both cytosol and membrane fractions of hypotonically lysed erythrocytes. We found that cytosolic Rho bound specifically to the cytoplasmic surface of the erythrocyte membrane and that the translocation of Rho to the membrane was absolutely dependent on the prior incubation of the cytosol with guanosine 5'--gamma-thio-triphosphate (1-50 microM) at low Mg2+ concentration. Rho binding sites could not be extracted from the membrane using conditions that extracted all other peripheral proteins and were unaffected by heat treatment and protease digestion. Rho binding was saturable, with a Kd in the range 1-5.0 nM, and the number of binding sites was estimated to be approx. (1-2) x 10(3) sites per cell. This is the first report of Rho binding to integral membrane components. The identity of these components may reveal novel aspects of the mechanism by which Rho exerts its multiple biochemical effects.

ADP Ribose Transferases↗
PubMed1997

The gene encoding protein 4.2 is distinct from the mouse platelet storage pool deficiency mutation pallid.

Previous studies identified the gene encoding the erythrocyte membrane protein 4.2 (Epb4.2) as a candidate for the mouse mutation pallid (pa); Epb4.2 genetically colocalized near pa on mouse Chromosome 2, and a truncated Epb4.2 transcript was present in tissues derived from pallid mice. We report here evidence that Epb4.2 and pa are not allelic. The pallid cDNA and intron/exon boundaries show no significant variation from the known BALB/c and C57BL/6J Epb4.2 sequence, and normal immunoreactive 72-kDa protein 4.2 is present in pallid tissues. Two recombinations between Epb4.2 and pa were identified in 173 phenotypically mutant (C57BL/6J-pa/pa x Mus castaneus) F2 animals. Northern blotting reveals a truncated Epb4.2 transcript in kidney mRNA from normal wild Mus domesticus (WSB/Ei) mice that comigrates with the pallid Epb4.2 mRNA. As the pa mutation originally arose in a wild M. domesticus mouse, we conclude that the Epb4.2 mRNA characteristic of pallid is a normal polymorphism derived from its wild ancestor and that Epb4.2 and pa are distinct loci.

Animals↗
PubMed1996

Role of N-myristylation in targeting of band 4.2 (pallidin) in nonerythroid cells.

Band 4.2 (pallidin) is a major erythrocyte membrane protein which has been detected in a number of nonerythroid cell types. Increasing evidence suggests that band 4.2 is involved in maintaining membrane stability in the erythrocyte. For example, band 4.2 binds to the integral membrane protein band 3 and to cytoskeletal proteins in the erythrocyte membrane, and band 4.2 deficiency results in varying degrees of hemolytic anemia. We have previously shown that human erythrocyte band 4.2 is myristylated at its penultimate glycine. Here we report that when expressed in both Sf9 and COS cells, myristylated forms of band 4.2 are detected at different intracellular locations than nonmyristylated forms. We also show that the unspliced form of human erythrocyte band 4.2 (a minor form in reticulocytes which contains an additional 30 amino acids after the first three N-terminal amino acids compared to the major erythroid form) is myristylated only at a barely detectable level, while mouse erythrocyte band 4.2 (homologous to the major erythroid form of human band 4.2) is myristylated at a level comparable to that of human band 4.2. These results suggest that myristylation plays a key role in the targeting of band 4.2 to specific intracellular locations and is likely to have a role in the function of this protein.

Animals↗
PubMed1996

Specific loss of protein kinase activities in senescent erythrocytes.

Rabbit erythrocytes of progressively increasing age were isolated using an avidin-biotin affinity technique and the activity of protein kinases and other enzymes was analyzed in cytosols and membranes from the isolated cells. The activities of cytosolic protein kinase C (PKC), cAMP-dependent kinase (PKA), and casein kinase type I and II (CKI and II) were all found to undergo an age-dependent decrease of twofold to fourfold over the 8-week lifespan of the cells. Membrane-associated tyrosine kinase showed little or no decrease, but membrane-associated CKI showed a dramatic eightfold decrease over the 8-week period. By contrast, various cytosolic enzymes, including lactate dehydrogenase, phosphoglycerate kinase, pyruvate kinase, and acid phosphatase, showed no change in activity over the same time period. Density-separated human erythrocytes showed qualitatively similar decreases in cytosolic protein kinase activities in the densest fractions, which contain the oldest cells. Our results show that aging erythrocytes undergo progressive loss of protein kinases that may adversely affect various cellular processes. The age-dependent loss of kinase activity reported here is one of the most striking manifestations of erythrocyte senescence yet to be reported.

Acid Phosphatase↗
PubMed1996

Human erythrocyte dematin and protein 4.2 (pallidin) are ATP binding proteins.

Dematin and protein 4.2 are peripheral membrane proteins associated with the cytoplasmic surface of the human erythrocyte plasma membrane. Isoforms of dematin and protein 4.2 exist in many nonerythroid cells. In solution, dematin is a trimeric protein containing two subunits of 48 kDa and one subunit of 52 kDa. Recent determination of the primary structure of the 52 kDa subunit of dematin showed that it contains an additional 22-amino acid sequence in the headpiece domain. An alignment of the 22-amino acid insertion sequence revealed that the 52 kDa subunit of dematin shares a novel 11-amino acid motif with protein 4.2. In this communication, we report that the conserved 11-amino acid motif in dematin52 and protein 4.2 contains a nucleotide binding P-loop. Direct binding of ATP is demonstrated to the glutathione S-transferase fusion proteins containing corresponding segments of dematin52 and protein 4.2 as well as to purified protein 4.2. The binding of ATP to the recombinant domains of dematin52 and protein 4.2 is specific, saturable, and of high affinity. The nucleotide specificity of the P-loop is restricted to ATP since no detectable binding was observed with GTP. These results show that the 11-amino acid motif provides an ATP binding site in dematin52 and protein 4.2. Although the functional significance of ATP binding is not yet clear, our findings open new perspectives for the function of dematin and protein 4.2 in vivo.

Adenosine Triphosphate↗
PubMed1996

Control of band 3 lateral and rotational mobility by band 4.2 in intact erythrocytes: release of band 3 oligomers from low-affinity binding sites.

Band 4.2 is a human erythrocyte membrane protein of incompletely characterized structure and function. Erythrocytes deficient in band 4.2 protein were used to examine the functional role of band 4.2 in intact erythrocyte membranes. Both the lateral and the rotational mobilities of band 3 were increased in band 4.2-deficient erythrocytes compared to control cells. In contrast, the lateral mobility of neither glycophorins nor a fluorescent phospholipid analog was altered in band 4.2-deficient cells. Compared to controls, band 4.2-deficient erythrocytes manifested a decreased ratio of band 3 to spectrin, and band 4.2-deficient membrane skeletons had decreased extractability of band 3 under low-salt conditions. Normal band 4.2 was found to bind to spectrin in solution and to promote the binding of spectrin to ankyrin-stripped inside-out vesicles. We conclude that band 4.2 provides low-affinity binding sites for both band 3 oligomers and spectrin dimers on the human erythrocyte membrane. Band 4.2 may serve as an accessory linking protein between the membrane skeleton and the overlying lipid bilayer.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1995

Phosphatidylinositol 3-kinase activity in murine erythroleukemia cells during DMSO-induced differentiation.

We have used murine erythroleukemia cells (MEL cells) to investigate the role of phosphatidylinositol 3-kinase (PI 3-kinase) in erythroid differentiation. When treated with dimethyl sulfoxide (DMSO), MEL cells grown on a fibronectin matrix become committed to erythroid differentiation asynchronously, with 90% of cells becoming committed by Day 3 of treatment. We found that during the first 3 days of DMSO treatment MEL cells showed a twofold increase in total PI 3-kinase activity and a fourfold increase in the highly phosphorylated PI 3-kinase product, PIP3. At the same time there was no change in the content of p85, the PI 3-kinase regulatory subunit. After Day 3, PI 3-kinase activity declined, in parallel with a disappearance of p85 antigen from the cells. Inclusion of the PI 3-kinase inhibitor Wortmannin in the culture medium resulted in an inhibition of cellular PI 3-kinase activity and a delay in DMSO-induced erythroid differentiation. These data suggest that PI 3-kinase may play a critical role during commitment of MEL cells to erythroid differentiation.

Androstadienes↗
PubMed1995

A point mutation in the protein 4.2 gene (allele 4.2 Tozeur) associated with hereditary haemolytic anaemia.

A recessively transmitted haemolytic anaemia associated with the lack of protein 4.2 was found in a Tunisian kindred. Trace amounts of this protein (72 kD component) became visible using high-sensitivity Western blots. Band 3 and ankyrin genes were excluded as candidate genes by linkage studies, and nucleotide sequencing of band 3 cytoplasmic domain cDNA revealed no alteration. In contrast, protein 4.2 gene contained in the homozygous state a mutation at position 310: CGA-->CAA (Arg-->Gln). This mutation defining allele 4.2 Tozeur was co-inherited with the disease. The mRNA encoding the variant protein was normal in size and approximately normal in amount. Recombinant protein 4.2 Tozeur bound normally to red cell IOVs but disclosed an increased susceptibility to proteolysis in vitro. We infer that the nearly total absence of protein 4.2 in the patients results from imbalance between destruction and synthesis of mutated protein 4.2 prior to its binding to the membrane.

Anemia, Hemolytic↗
PubMed1994

Evidence for the association of protein 4.1 immunoreactive forms with neurofibrillary tangles in Alzheimer's disease brains.

The formation of neurofibrillary tangles (NFTs) and paired-helical filaments (PHFs) in Alzheimer's disease (AD) reflects a major disorganization of the cytoskeleton. The role of the neuronal membrane skeleton in the development of these abnormalities has not previously been investigated. In this study, we used 9 antibodies raised against the erythrocyte membrane skeleton protein 4.1 (P4.1) for immunocytochemical and immunoblot analyses to investigate whether or not the brain homologues of this protein were constituents of NFTs or PHFs. Our results show that 7 of the 9 monospecific antibodies against the human and pig erythrocyte P4.1 stained NFTs in the prefrontal cortex and hippocampus of AD brains. The P4.1 antibodies used here did not cross-react with tau protein isolated from AD brain, and preabsorption of these antibodies with tau protein did not cause loss of NFT staining. In age-matched control brains, these P4.1 antibodies stained neuronal cell bodies or nuclei. Six of the antibodies also stained isolated NFTs but the SDS-insoluble NFTs were immunostained only by two of the P4.1 antibodies. By using inositol hexaphosphate affinity chromatography and immunoblot analysis, we identified a 68-kDa protein as the most likely brain analogue of P4.1. When SDS-extracted proteins from the isolated NFTs were immunoblotted, a 50-kDa band was immunostained. The 68-kDa and 50-kDa proteins were not stained by tau protein and neurofilament subunit NF-H antibodies, that strongly stained NFTs. We conclude that brain protein 4.1 isoform(s) are constituents of NFTs in AD.

Aged↗
PubMed1994

Absence of high-affinity band 4.1 binding sites from membranes of glycophorin C- and D-deficient (Leach phenotype) erythrocytes.

We investigated the role of glycophorins C and D in the association of band 4.1 with the erythrocyte membrane by measuring the binding of band 4.1 to erythrocyte inside-out vesicles stripped of endogenous band 4.1. Vesicles were prepared from either normal erythrocytes or erythrocytes completely lacking glycophorins C and D (Leach phenotype). Band 4.1 binding to vesicles from normal erythrocytes gave rise to a nonlinear Scatchard plot, indicative of two classes of binding sites: a low-capacity, high-affinity class of sites (about 10% of the total) and a high-capacity, low-affinity class of sites. Vesicles prepared from Leach erythrocytes had a binding capacity for band 4.1 that was, on average, 32% lower than that of vesicles from normal erythrocytes. This difference was caused by the complete absence of the high-affinity binding sites as well as by a decrease in the number of low-affinity binding sites. Reduction of membrane phosphatidylinositol 4,5-biphosphate (PIP2) content by adenosine triphosphate depletion or activation of phosphoinositidase C resulted in a decrease in band 4.1 binding capacity to a similar extent in both control and Leach vesicles. The principal effect of PIP2 depletion was a reduction in the number of low-affinity band 4.1 binding sites in control and Leach vesicles. The fact that PIP2 depletion induced a decrease in band 4.1 binding to Leach vesicles shows that glycophorin C or D is not required for the formation of PIP2-sensitive band 4.1 binding sites, and may not be involved in PIP2-sensitive band 4.1 binding sites even when they are present. Our studies give new insights into the involvement of glycophorins and of PIP2 in modulating cytoskeletal-membrane interactions.

Adenosine Triphosphate↗
PubMed1994

cDNA sequence, gene sequence, and properties of murine pallidin (band 4.2), the protein implicated in the murine pallid mutation.

Band 4.2, which plays an important but poorly understood role in erythrocyte function and survival, is a major component of erythrocyte membranes. Recently, it has been shown that the gene for murine protein band 4.2 colocalizes on chromosome 2 with the murine pallid mutation, which affects the formation or function of intracellular storage granules in melanocytes and platelets and lysosomes in kidney. As a first step in identifying the mutation responsible for the pallid phenotype, we have sequenced the entire normal murine band 4.2 gene. Our results show that the gene for murine band 4.2 is approximately 22 kb in size, with 13 exons and 12 intervening introns. The organization of the mouse band 4.2 gene is identical to that of the human band 4.2 gene and similar to that of the genes for the transglutaminase enzymes, reiterating the membership of protein band 4.2 in the transglutaminase gene superfamily. We also present 3.5 kb of normal murine erythroid band 4.2 cDNA sequence containing an open reading frame of 2073 bp and coding for 691 amino acids. This is the same size as the human erythrocyte protein, with which the murine protein shares a 72% amino acid identity.

Amino Acid Sequence↗
PubMed1993

Phorbol 12-myristate 13-acetate-stimulated phosphorylation of erythrocyte membrane skeletal proteins is blocked by calpain inhibitors: possible role of protein kinase M.

Human erythrocytes contain cytosolic protein kinase C (PKC) which, when activated by phorbol 12-myristate 13-acetate (PMA), induces the phosphorylation of the membrane skeletal proteins band 4.1, band 4.9 and adducin. We found that brief treatments of erythrocytes with PMA resulted in a decrease in cytosolic PKC content and in the transient appearance in the cytosol of a Ca(2+)- and phospholipid-independent 55 kDa fragment of PKC, called PKM. Prolonged treatment with PMA resulted in the complete and irreversible loss of erythrocyte PKC. To investigate the possible role of calpain in this process, the calpain inhibitors leupeptin and E-64 were sealed inside erythrocytes by reversible haemolysis. Both inhibitors prolonged the lifetime of PKC in PMA-treated cells, and leupeptin was shown to block the PMA-stimulated appearance of PKM in the cytosol. Significantly, leupeptin also completely blocked PMA-stimulated phosphorylation of membrane and cytosolic substrates. This effect was mimicked by other calpain inhibitors (MDL-28170 and calpain inhibitor I), but did not occur when other protease inhibitors such as phenylmethanesulphonyl fluoride, pepstatin A or chymostatin were used. In addition, the phosphorylation of exogenous histone sealed inside erythrocytes was also blocked by leupeptin. Immunoblotting showed that leupeptin did not prevent the PMA-induced translocation of PKC to the erythrocyte membrane. Thus inhibition of PKC phosphorylation of membrane skeletal proteins by calpain inhibitors was not due to inhibition of PKC translocation to the membrane. Our results suggest that PMA treatment of erythrocytes results in the translocation of PKC to the plasma membrane, followed by calpain-mediated cleavage of PKC to PKM. This cleavage, or some other leupeptin-inhibitable process, is a necessary step for the phosphorylation of membrane skeletal substrates, suggesting that the short-lived PKM may be responsible for membrane skeletal phosphorylation. Our results suggest a potential mechanism whereby erythrocyte PKC may be subject to continual down-regulation during the lifespan of the erythrocyte due to repeated activation events, possibly related to transient Ca2+ influx. Such down-regulation may play an important role in erythrocyte survival or pathophysiology.

Amino Acid Sequence↗
PubMed1993

Structural domain mapping and phosphorylation of human erythrocyte pallidin (band 4.2).

Pallidin (band 4.2) is a major protein of the human erythrocyte membrane, and plays an important but as yet undefined role in maintaining the normal shape and lifespan of the erythrocyte. The pallidin protein has been purified by a new procedure which yields a protein which is > 97% pure as judged by gel electrophoresis, while pallidin purified by our original procedure is only approx. 85% pure. The new form of the protein is unstable in physiological salt solutions. However, taking advantage of its high purity, we have used the new form of the protein to produce a structural domain map of its principal tryptic fragments. We also show that pallidin can be phosphorylated by a red-cell membrane kinase which partially co-purifies with it, and has properties similar to the catalytic subunit of cAMP-dependent kinase. Both cAMP-dependent kinase and the red-cell kinase phosphorylate the same tryptic domains on the pallidin protein. Our results show that endogenous pallidin on the red-cell membrane is a poor substrate for the kinase, possibly because it is fully phosphorylated, or inaccessible to the kinase.

Actins↗
PubMed1993

The role of inositol phospholipids in the association of band 4.1 with the human erythrocyte membrane.

Band 4.1 is a major protein of the erythrocyte membrane skeleton. It promotes the binding of spectrin to F-actin and may anchor the skeletal network to the plasma membrane via its association with integral membrane proteins. Here, we have investigated the involvement of inositol phospholipids in the binding of band 4.1 to erythrocyte membranes using membrane vesicles stripped of all peripheral proteins at alkaline pH. Trypsinization of these vesicles allows the discrimination of two classes of band 4.1 binding sites: trypsin-sensitive sites (60-65% of the total), largely or exclusively on band 3, and trypsin-resistant sites (35-40% of the total), composed, at least in part, of the glycophorins. ATP depletion or activation of erythrocyte phosphoinositol phospholipase C led to a reduction in membrane phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] content by 20-70% in different experiments. The resulting decrease of band 4.1 binding to vesicles by was variable, but averaged about 15-20%. The same treatments led to an average decrease in the band 4.1 binding capacity of trypsinized vesicles of 55%. Since this is equivalent to a 20% decrease in the binding capacity of non-trypsinized vesicles (consistent with the above result), it indicates that PtdIns(4,5)P2 regulates the binding of band 4.1 only to trypsin-resistant binding sites (and to only a subset of these) accounting for about 15-20% of total band 4.1 binding sites on membranes. We found that hydrolysis of > 95% of PtdIns(4,5)P2 with exogenous phospholipase C-delta (PLC delta) resulted in no further decrease in band 4.1 binding to vesicles than did hydrolysis of 65-70% of PtdIns(4,5)P2 which is accessible to erythrocyte phosphoinositol phospholipase C. This suggests that only 65-70% of total membrane PtdIns(4,5)P2 is involved in regulating band 4.1 binding. Significantly, the pool of PtdIns(4,5)P2 involved is the same pool which can be hydrolysed by erythrocyte phosphoinositol phospholipase C, and which has been shown to be metabolically labile in erythrocytes. The membrane binding capacity for band 4.1 found in this study (averaging 1000 micrograms/mg vesicle protein) is considerably higher than that found in previous studies. The results are consistent with the existence of a binding site for band 4.1 on each copy of the major transmembrane proteins (band 3 and the glycophorins). These results provide new insights into the involvement of membrane inositol phospholipids in cytoskeletal-membrane interactions.

Adenosine Triphosphate↗