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Jon S Morrow

Publications and source records attributed to Jon S Morrow.

15 recordsLinked to original sources
PubMed2007

Sequential degradation of alphaII and betaII spectrin by calpain in glutamate or maitotoxin-stimulated cells.

Calpain-catalyzed proteolysis of II-spectrin is a regulated event associated with neuronal long-term potentiation, platelet and leukocyte activation, and other processes. Calpain proteolysis is also linked to apoptotic and nonapoptotic cell death following excessive glutamate exposure, hypoxia, HIV-gp120/160 exposure, or toxic injury. The molecular basis for these divergent consequences of calpain action, and their relationship to spectrin proteolysis, is unclear. Calpain preferentially cleaves II spectrin in vitro in repeat 11 between residues Y1176 and G1177. Unless stimulated by Ca++ and calmodulin (CaM), betaII spectrin proteolysis in vitro is much slower. We identify additional unrecognized sites in spectrin targeted by calpain in vitro and in vivo. Bound CaM induces a second II spectrin cleavage at G1230*S1231. BetaII spectrin is cleaved at four sites. One cleavage only occurs in the absence of CaM at high enzyme-to-substrate ratios near the betaII spectrin COOH-terminus. CaM promotes II spectrin cleavages at Q1440*S1441, S1447*Q1448, and L1482*A1483. These sites are also cleaved in the absence of CaM in recombinant II spectrin fusion peptides, indicating that they are probably shielded in the spectrin heterotetramer and become exposed only after CaM binds alphaII spectrin. Using epitope-specific antibodies prepared to the calpain cleavage sites in both alphaII and betaII spectrin, we find in cultured rat cortical neurons that brief glutamate exposure (a physiologic ligand) rapidly stimulates alphaII spectrin cleavage only at Y1176*G1177, while II spectrin remains intact. In cultured SH-SY5Y cells that lack an NMDA receptor, glutamate is without effect. Conversely, when stimulated by calcium influx (via maitotoxin), there is rapid and sequential cleavage of alphaII and then betaII spectrin, coinciding with the onset of nonapoptotic cell death. These results identify (i) novel calpain target sites in both alphaII and betaII spectrin; (ii) trans-regulation of proteolytic susceptibility between the spectrin subunits in vivo; and (iii) the preferential cleavage of alphaII spectrin vs betaII spectrin when responsive cells are stimulated by engagement of the NMDA receptor. We postulate that calpain proteolysis of spectrin can activate two physiologically distinct responses: one that enhances skeletal plasticity without destroying the spectrin-actin skeleton, characterized by preservation of betaII spectrin; or an alternative response closely correlated with nonapoptotic cell death and characterized by proteolysis of betaII spectrin and complete dissolution of the spectrin skeleton.

Amino Acid Sequence↗
PubMed2006

Structure of the calmodulin alphaII-spectrin complex provides insight into the regulation of cell plasticity.

AlphaII-spectrin is a major cortical cytoskeletal protein contributing to membrane organization and integrity. The Ca2+-activated binding of calmodulin to an unstructured insert in the 11th repeat unit of alphaII-spectrin enhances the susceptibility of spectrin to calpain cleavage but abolishes its sensitivity to several caspases and to at least one bacterially derived pathologic protease. Other regulatory inputs including phosphorylation by c-Src also modulate the proteolytic susceptibility of alphaII-spectrin. These pathways, acting through spectrin, appear to control membrane plasticity and integrity in several cell types. To provide a structural basis for understanding these crucial biological events, we have solved the crystal structure of a complex between bovine calmodulin and the calmodulin-binding domain of human alphaII-spectrin (Protein Data Bank ID code 2FOT). The structure revealed that the entire calmodulin-spectrin-binding interface is hydrophobic in nature. The spectrin domain is also unique in folding into an amphiphilic helix once positioned within the calmodulin-binding groove. The structure of this complex provides insight into the mechanisms by which calmodulin, calpain, caspase, and tyrosine phosphorylation act on spectrin to regulate essential cellular processes.

Amino Acid Sequence↗
PubMed2006

Stimulation of Galphaq-coupled M1 muscarinic receptor causes reversible spectrin redistribution mediated by PLC, PKC and ROCK.

Spectrin is a cytoskeletal protein that plays a role in formation of the specialized plasma membrane domains. However, little is known of the molecular mechanism that regulates responses of spectrin to extracellular stimuli, such as activation of G-protein-coupled receptor (GPCR). We have found that alphaII spectrin is a component of the Galpha(q/11)-associated protein complex in CHO cells stably expressing the M1 muscarinic receptor, and investigated the effect of activation of GPCR on the cellular localization of yellow-fluorescent-protein-tagged alphaII spectrin. Stimulation of Galpha(q/11)-coupled M1 muscarinic receptor triggered reversible redistribution of alphaII spectrin following a rise in intracellular Ca2+ concentration. This redistribution, accompanied by non-apoptotic membrane blebbing, required an intact actin cytoskeleton and was dependent on activation of phospholipase C, protein kinase C, and Rho-associated kinase ROCK. Muscarinic-agonist-induced spectrin remodeling appeared particularly active at localized domains, which is clear contrast to that caused by constitutive activation of ROCK and to global rearrangement of the spectrin lattice caused by changes in osmotic pressure. These results suggest a role for spectrin in providing a dynamic and reversible signaling platform to the specific domains of the plasma membrane in response to stimulation of GPCR.

Animals↗
PubMed2006

Human Sec31B: a family of new mammalian orthologues of yeast Sec31p that associate with the COPII coat.

We have cloned human brain and testis Sec31B protein (also known as secretory pathway component Sec31B-1 or SEC31-like 2; GenBank accession number AF274863). Sec31B is an orthologue of Saccharomyces cerevisiae Sec31p, a component of the COPII vesicle coat that mediates vesicular traffic from the endoplasmic reticulum. Sec31B is widely expressed and enriched in cerebellum and testis. Its predicted sequence of 1180 residues (expected molecular mass 128,711 Da) shares 47.3% and 18.8% similarity to human Sec31A (also known as Sec31; GenBank accession number AF139184) and yeast Sec31p, respectively. The gene encoding Sec31B is located on chromosome 10q24 and contains 29 exons. PCR analysis of exon utilization reveals massive alternative mRNA splicing of Sec31B, with just 16 exons being constitutively utilized in all transcripts. The presence of a stop codon in exon 13 generates two families of Sec31B gene products (each displaying additional patterns of mRNA splicing): a group of full-length proteins (hereafter referred to as Sec31B-F) and also a group of truncated proteins (hereafter referred to as Sec31B-T), distinguished by their utilization of exon 13. Sec31B-F closely resembles Sec31p and Sec31A, with canonical WD repeats in an N-terminal domain that binds Sec13 and a proline-rich C-terminal region that presumably binds Sec23/24. The Sec31B-T group (molecular mass 52,983 Da) contains a preserved WD-repeat domain but lacks the C-terminal proline-rich region. When expressed as a fusion protein with eYFP in cultured cells, Sec31B-F associates with the endoplasmic reticulum and with vesicular-tubular clusters, displays restricted intracellular movement characteristic of COPII vesicle dynamics, co-distributes on organelles with Sec13, Sec31A and Sec23 (markers of the COPII coat), and concentrates with ts045-VSV-G-CFP (VSV-G) when examined early in the secretory pathway or after temperature or nocodazole inhibition. The role of the truncated form Sec31B-T appears to be distinct from that of Sec31B-F and remains unknown. We conclude that Sec31B-F contributes to the diversity of the mammalian COPII coat, and speculate that the Sec31 cage, like Sec24, might be built with isoforms tuned to specific types of cargo or to other specialized functions.

Alternative Splicing↗
PubMed2006

Guidelines for interactions between clinical faculty and the pharmaceutical industry: one medical school's approach.

A productive and ethical relationship between the pharmaceutical industry and physicians is critical to improving drug discovery and public health. In response to concerns about inappropriate financial relationships between the pharmaceutical industry and physicians, national organizations representing physicians or industry have made recommendations designed to reduce conflicts of interest, legal exposure, and dissemination of biased information. Despite these initiatives, the prescribing practices of physicians may be unduly influenced by the marketing efforts of industry and physicians may inadvertently distribute information that is biased in favor of a commercial entity. Moreover, physicians may be vulnerable to prosecution through federal anti-kickback and false claims statutes because of potentially inappropriate financial relationships with pharmaceutical companies. Since academic medical centers have a critical role in establishing professional standards, the faculty of Yale University School of Medicine developed guidelines for the relationships of faculty with the pharmaceutical industry, which were approved in May 2005. Input from clinical faculty and from representatives of the pharmaceutical industry was utilized in formulating the guidelines. In contrast to existing recommendations, the Yale guidelines, which are presented as an Appendix here, ban faculty from receiving any form of gift, meal, or free drug sample (for personal use) from industry, and set more stringent standards for the disclosure and resolution of financial conflict of interest in Yale's educational programs. The growing opportunities for drug discovery, the need to use medications in a more evidence-based manner, and preservation of the public trust require the highest professional standards of rigor and integrity. These guidelines are offered as part of the strategy to meet this compelling challenge.

Codes of Ethics↗
PubMed2004

Mutation of a highly conserved isoleucine disrupts hydrophobic interactions in the alpha beta spectrin self-association binding site.

We studied an infant with severe neonatal hemolytic anemia and hyperbilirubinemia that evolved into a partially compensated ellipto-poikilocytic anemia. His father had typical elliptocytosis. Their erythrocyte membranes demonstrated structural and functional defects in spectrin. Genetic studies revealed that the proband and his father were heterozygous for an alpha-spectrin mutation, Ile24Thr, in the alpha beta spectrin self-association binding site. The proband also carried the low expression allele alpha(LELY) in trans, influencing the clinical phenotype. The importance of isoleucine in this position of the proposed triple helical model of spectrin repeats is highlighted by its evolutionary conservation in all alpha spectrins from Drosophila to humans. Molecular modeling demonstrated that replacement of a hydrophobic isoleucine with a hydrophilic threonine disrupts highly conserved hydrophobic interactions in the interior of the spectrin triple helix critical for spectrin function.

Binding Sites↗
PubMed2003

Identification of a stomatin orthologue in vacuoles induced in human erythrocytes by malaria parasites. A role for microbial raft proteins in apicomplexan vacuole biogenesis.

When the human malaria parasite Plasmodium falciparum infects erythrocytes, proteins associated with host-derived detergent-resistant membrane (DRM) rafts are selectively recruited into the newly formed vacuole, but parasite proteins that contribute to raft-based vacuole development are unknown. In mammalian cells, DRM-associated integral membrane proteins such as caveolin-1 and flotillin-1 that form oligomers have been linked to the formation of DRM-based invaginations called caveolae. Here we show that the P. falciparum genome does not encode caveolins or flotillins but does contain an orthologue of human band 7 stomatin, a protein known to oligomerize, associate with non-caveolar DRMs and is distantly related to flotillins. Stomatins are members of a large protein family conserved in evolution and P. falciparum (Pf) stomatin appears to be a prokaryotic-like molecule. Evidence is presented that it associates with DRMs and may oligomerize, suggesting that these features are conserved in the stomatin family. Further, Pfstomatin is an integral membrane protein concentrated at the apical end of extracellular parasites, where it co-localizes with invasion-associated rhoptry organelles. A resident rhoptry protein, RhopH2 also resides in DRMs. This provides the first evidence that rhoptries of an apicomplexan parasite contain DRM rafts. Further, when the parasite invades erythrocytes, rhoptry Pfstomatin and RhopH2 are inserted into the newly formed vacuole. Thus, like caveolin-1 and flotillin-1, a stomatin may also associate with non-clathrin coated, DRM-enriched vacuoles. We propose a new model of invasion and vacuole formation involving DRM-based interactions of both host and parasite molecules.

Amino Acid Sequence↗
PubMed2003

Neural cell adhesion molecule (NCAM) association with PKCbeta2 via betaI spectrin is implicated in NCAM-mediated neurite outgrowth.

In hippocampal neurons and transfected CHO cells, neural cell adhesion molecule (NCAM) 120, NCAM140, and NCAM180 form Triton X-100-insoluble complexes with betaI spectrin. Heteromeric spectrin (alphaIbetaI) binds to the intracellular domain of NCAM180, and isolated spectrin subunits bind to both NCAM180 and NCAM140, as does the betaI spectrin fragment encompassing second and third spectrin repeats (betaI2-3). In NCAM120-transfected cells, betaI spectrin is detectable predominantly in lipid rafts. Treatment of cells with methyl-beta-cyclodextrin disrupts the NCAM120-spectrin complex, implicating lipid rafts as a platform linking NCAM120 and spectrin. NCAM140/NCAM180-betaI spectrin complexes do not depend on raft integrity and are located both in rafts and raft-free membrane domains. PKCbeta2 forms detergent-insoluble complexes with NCAM140/NCAM180 and spectrin. Activation of NCAM enhances the formation of NCAM140/NCAM180-spectrin-PKCbeta2 complexes and results in their redistribution to lipid rafts. The complex is disrupted by the expression of dominant-negative betaI2-3, which impairs binding of spectrin to NCAM, implicating spectrin as the bridge between PKCbeta2 and NCAM140 or NCAM180. Redistribution of PKCbeta2 to NCAM-spectrin complexes is also blocked by a specific fibroblast growth factor receptor inhibitor. Furthermore, transfection with betaI2-3 inhibits NCAM-induced neurite outgrowth, showing that formation of the NCAM-spectrin-PKCbeta2 complex is necessary for NCAM-mediated neurite outgrowth.

Animals↗
PubMed2002

c-Src binds alpha II spectrin's Src homology 3 (SH3) domain and blocks calpain susceptibility by phosphorylating Tyr1176.

Spectrin is a ubiquitous heterodimeric scaffolding protein that stabilizes membranes and organizes protein and lipid microdomains on both the plasma membrane and intracellular organelles. Phosphorylation of beta-spectrin on Ser/Thr is well recognized. Less clear is whether alpha-spectrin is phosphorylated in vivo and whether spectrin is phosphorylated on tyrosine (pTyr). We affirmatively answer both questions. In cultured Madin-Darby canine kidney cells, alphaII spectrin undergoes in vivo tyrosine phosphorylation. Enhancement of the steady state level of pTyr-modified alphaII spectrin by vanadate, a phosphatase inhibitor, implies a dynamic balance between alphaII spectrin phosphorylation and dephosphorylation. Recombinant peptides containing the Src homology 3 domain of alphaII spectrin (but not the Src homology 3 domain of alphaI spectrin) bind specifically to phosphorylated c-Src in Madin-Darby canine kidney cell lysates, suggesting that this kinase is responsible for its in vivo phosphorylation. pTyr-modified alphaII spectrin is resistant to maitotoxin-induced cleavage by mu-calpain in vivo. In vitro studies of recombinant alphaII spectrin peptides representing repeats 9-12 identify two sites of pTyr modification. The first site is at Tyr(1073), a residue immediately adjacent to a region encoded by alternative exon usage (insert 1). The second site is at Tyr(1176). This residue flanks the major site of cleavage by the calcium-dependent protease calpain, and phosphorylation of Tyr(1176) by c-Src reduces the susceptibility of alphaII spectrin to cleavage by mu-calpain. Calpain cleavage of spectrin, activated by Ca(2+) and calmodulin, contributes to diverse cellular processes including synaptic remodeling, receptor-mediated endocytosis, apoptosis, and the response of the renal epithelial cell to ischemic injury. Tyrosine phosphorylation of alphaII spectrin now would appear to also mediate these events. The spectrin skeleton thus forms a point of convergence between kinase/phosphatase and Ca(2+)-mediated signaling cascades.

Animals↗
PubMed2002

Disrupted synaptic development in the hypoxic newborn brain.

Infants born prematurely risk significant life-long cognitive disability, representing a major pediatric health crisis. The neuropathology of this cohort is accurately modeled in mice subjected to sublethal postnatal hypoxia. Massively parallel transcriptome analysis using cDNA microchips (9,262 genes), combined with immunohistochemical and protein assays, reveals that sublethal hypoxia accentuates genes subserving presynaptic function, and it suppresses genes involved with synaptic maturation, postsynaptic function, and neurotransmission. Other significantly affected pathways include those involved with glial maturation, vasculogenesis, and components of the cortical and microtubular cytoskeleton. These patterns reveal a global dysynchrony in the maturation programs of the hypoxic developing brain, and offer insights into the vulnerabilities of processes that guide early postnatal cerebral maturation.

Animals↗
PubMed2002

Fragmentation of the Golgi apparatus. A role for beta III spectrin and synthesis of phosphatidylinositol 4,5-bisphosphate.

Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) synthesis has been implicated in maintaining the function of the Golgi apparatus. Here we demonstrate that the inhibition of PtdIns(4,5)P(2) synthesis in vitro in response to primary alcohol treatment and the kinetics of Golgi fragmentation in vivo were very rapid and tightly coupled. Preloading Golgi membranes with short chain phosphatidic acid abrogated the alcohol-mediated inhibition of PtdIns(4,5)P(2) synthesis in vitro. We also show that fragmentation of the Golgi apparatus in response to diminished PtdIns(4,5)P(2) synthesis correlated with both the phosphorylation of a Golgi form of beta III spectrin, a PtdIns(4,5)P(2)-interacting protein, and changes in its intracellular redistribution. The data are consistent with a model suggesting that the decreased PtdIns(4,5)P(2) synthesis and the phosphorylation state of beta III spectrin modulate the structural integrity of the Golgi apparatus.

Animals↗
PubMed2002

Dissociation of spectrin-ankyrin complex as a basis for loss of Na-K-ATPase polarity after ischemia.

The polarized distribution of Na-K-ATPase at the basolateral membranes of renal tubule epithelial cells is maintained via a tethering interaction with the underlying spectrin-ankyrin cytoskeleton. In this study, we have explored the mechanism underlying the loss of Na-K-ATPase polarity after ischemic injury in Madin-Darby canine kidney (MDCK) cells, utilizing a novel antibody raised against a recently described kidney-specific isoform of ankyrin. In control MDCK cells, ankyrin was colocalized with Na-K-ATPase at the basolateral membrane. ATP depletion resulted in a duration-dependent mislocation of Na-K-ATPase and ankyrin throughout the cytoplasm. Colocalization studies showed a partial overlap between the distribution of ankyrin and Na-K-ATPase at all periods after ATP depletion. By immunoprecipitation with anti-ankyrin antibody, the mislocated Na-K-ATPase remained bound to ankyrin at all time points after ATP depletion. However, the interaction between ankyrin and spectrin was markedly diminished within 3 h of ATP depletion and was completely lost after 6 h. In solution binding assays using a fusion peptide of glutathione S-transferase with the ankyrin binding domain of Na-K-ATPase, a complex with ankyrin was detected at all time points after ATP depletion, but spectrin was lost from the complex in a duration-dependent manner. The loss of spectrin binding was not attributable to spectrin degradation but was associated with hyperphosphorylation of ankyrin. The results suggest that a dissociation of the membrane-cytoskeleton complex at the spectrin-ankyrin interface may contribute to the loss of Na-K-ATPase polarity after ischemic injury and reaffirm a critical adapter role for ankyrin in the normal maintenance of Na-K-ATPase polarity.

Adenosine Triphosphate↗
PubMed2002

Rad9 phosphorylation sites couple Rad53 to the Saccharomyces cerevisiae DNA damage checkpoint.

Rad9 is required for the MEC1/TEL1-dependent activation of Saccharomyces cerevisiae DNA damage checkpoint pathways mediated by Rad53 and Chk1. DNA damage induces Rad9 phosphorylation, and Rad53 specifically associates with phosphorylated Rad9. We report here that multiple Mec1/Tel1 consensus [S/T]Q sites within Rad9 are phosphorylated in response to DNA damage. These Rad9 phosphorylation sites are selectively required for activation of the Rad53 branch of the checkpoint pathway. Consistent with the in vivo function in recruiting Rad53, Rad9 phosphopeptides are bound by Rad53 forkhead-associated (FHA) domains in vitro. These data suggest that functionally independent domains within Rad9 regulate Rad53 and Chk1, and support the model that FHA domain-mediated recognition of Rad9 phosphopeptides couples Rad53 to the DNA damage checkpoint pathway.

Binding Sites↗
PubMedDate not supplied

Antibodies to betaISigma2 spectrin identify in-homogeneities in the erythrocyte membrane skeleton.

The cortical cytoskeleton of the mammalian red cell, composed of spectrin, actin, protein 4.1, adducin, and protein 4.9, is generally regarded as a homogeneous structure that maintains the integrity of the membrane and the lateral disposition of integral membrane proteins. The major component of this structure is a hetero-oligomer of alphaI and betaISigma1 spectrin. In other tissues, most notably muscle and brain, a transcript of the betaI spectrin gene is generated by alternative exon utilization, yielding a protein that has the COOH-terminal 19 residues of betaISigma1 spectrin replaced by 210 novel residues to generate betaISigma2 spectrin. This new transcript contains a pleckstrin homology (PH) domain and may even exist under some conditions in a homopolymeric form. Using antibodies specific for the COOH-terminal domains of either betaISigma1 or betaISigma2 spectrin, we find that contrary to previous understandings, mature human erythrocytes contain a subpopulation of spectrin that is immunoreactive with antibodies to the betaISigma2 isoform, and that this spectrin is distributed into distinct plasma membrane patches. These results suggest that the native mammalian erythrocyte membrane skeleton, rather than being homogeneous, contains discrete submicron-scale microdomains that differ in both their composition and dispersion across the cell surface. The precise nature and role of these putative microdomains is under active investigation.

Antibodies, Monoclonal↗