Anthony C. Kim

THE PDZ DOMAIN OF HUMAN ERYTHROCYTE P55 MEDIATES ITS BINDING TO THE CYTOPLASMIC CARBOXYL TERMINUS OF GLYCOPHORIN C: ANALYSIS OF THE BINDING INTERFACE BY IN VITRO MUTAGENESIS

The PDZ domain, also known as the GLGF repeat/DHR domain, is an ∼90-amino acid motif discovered in a recently identified family of proteins termed MAGUKs (membrane-associated guanylatekinase homologues). Sequence comparison analysis has since identified PDZ domains in over 50 proteins. Like SH2 and SH3 domains, the PDZ domains mediate specific protein-protein interactions, whose specificities appear to be dictated by the primary structure of the PDZ domain as well as its binding target. Using recombinant fusion proteins and a blot overlay assay, we show that a single copy of the PDZ domain in human erythrocyte p55 binds to the carboxyl terminus of the cytoplasmic domain of human erythroid glycophorin C. Deletion mutagenesis of 21 amino acids at the amino terminus of the p55 PDZ domain completely abrogates its binding activity for glycophorin C. Using an alanine scan and surface plasmon resonance technique, we identify residues in the cytoplasmic domain of glycophorin C that are critical for its interaction with the PDZ domain. The recognition specificity of the p55 PDZ domain appears to be unique, since the three PDZ domains of hDlg (human lymphocyte homologue of the Drosophiladiscs large tumor suppressor) do not bind the cytoplasmic domain of glycophorin C. Taken together with our previous studies, these results complete the identification of interacting domains in the ternary complex between p55, glycophorin C, and protein 4.1. Implications of these findings are discussed in terms of binding specificity and the regulation of cytoskeleton-membrane interactions.

Headpiece domain of dematin is required for the stability of the erythrocyte membrane

Dematin is an actin-binding and bundling protein of the erythrocyte membrane skeleton. Dematin is localized to the spectrin–actin junctions, and its actin-bundling activity is regulated by phosphorylation of cAMP-dependent protein kinase. The carboxyl terminus of dematin is homologous to the “headpiece” domain of villin, an actin-bundling protein of the microvillus cytoskeleton. The headpiece domain contains an actin-binding site, a cAMP-kinase phosphorylation site, plays an essential role in dematin self-assembly, and bundles F-actin in vitro. By using homologous recombination in mouse embryonic stem cells, the headpiece domain of dematin was deleted to evaluate its function in vivo. Dematin headpiece null mice were viable and born at the expected Mendelian ratio. Hematological evaluation revealed evidence of compensated anemia and spherocytosis in the dematin headpiece null mice. The headpiece null erythrocytes were osmotically fragile, and ektacytometry/micropore filtration measurements demonstrated reduced deformability and filterability. In vitro membrane stability measurements indicated significantly greater membrane fragmentation of the dematin headpiece null erythrocytes. Finally, biochemical characterization, including the vesicle/cytoskeleton dissociation, spectrin self-association, and chemical crosslinking measurements, revealed a weakened membrane skeleton evidenced by reduced association of spectrin and actin to the plasma membrane. Together, these results provide evidence for the physiological significance of dematin and demonstrate a role for the headpiece domain in the maintenance of structural integrity and mechanical properties of erythrocytes in vivo.

Reassignment of the EPB4·1 gene to 1p36 and assessment of its involvement in neuroblastomas

Objectives EPB4·1 has been previously mapped to human chromosome 1p33‐p34.2. In contradiction to this chromosomal location, we have mapped EPB4·1–1p36 by using fluorescence in situ hybridization and radiation hybrid mapping. In neuroblastomas, deletions of the telomeric end of chromosome 1 (1p36) are the most common genetic aberration.

Erythrocyte scaffolding protein p55/MPP1 functions as an essential regulator of neutrophil polarity

As mediators of innate immunity, neutrophils respond to chemoattractants by adopting a highly polarized morphology. Efficient chemotaxis requires the formation of one prominent pseudopod at the cell front characterized by actin polymerization, while local inhibition suppresses the formation of rear and lateral protrusions. This asymmetric control of signaling pathways is required for directional migration along a chemotactic gradient. Here, we identify the MAGUK protein p55/MPP1 as a mediator of the frontness signal required for neutrophil polarization. We developed a p55 knockout (p55−/−) mouse model, and demonstrate that p55−/− neutrophils form multiple transient pseudopods upon chemotactic stimulation, and do not migrate efficiently in vitro. Upon agonist stimulation, p55 is rapidly recruited to the leading edge of neutrophils in mice and humans. Total F-actin polymerization, along with Rac1 and RhoA activation, appear to be normal in p55−/− neutrophils. Importantly, phosphorylation of Akt is significantly decreased in p55−/− neutrophils upon chemotactic stimulation. The activity of immunoprecipitated phosphatidylinositol 3-kinase γ (PI3Kγ), responsible for chemoattractant-induced synthesis of PIP3 and Akt phosphorylation, is unperturbed in p55−/− neutrophils. Although the total amount of PIP3 is normal in p55−/− neutrophils, PIP3 is diffusely localized and forms punctate aggregates in activated p55−/− neutrophils, as compared to its accumulation at the leading edge membrane in the wild type neutrophils. Together, these results show that p55 is required for neutrophil polarization by regulating Akt phosphorylation through a mechanism that is independent of PI3Kγ activity.

CDNA SEQUENCE, GENOMIC STRUCTURE, AND EXPRESSION OF THE MOUSE DEMATIN GENE(EPB4.9)

Dematin (protein 4.9 region) is an actin-bundling phosphoprotein of the erythroid membrane cytoskeleton (Rana et al. 1993; Azim et al. 1995). Although much is known about the functions of the core group of erythroid membrane proteins, such as spectrin, ankyrin, band 3, and protein 4.1, relatively little is known concerning the physiological role of dematin in vivo. In vitro, dematin’s actinbundling activity is abolished upon phosphorylation by the cAMPdependent protein kinase (Chishti et al. 1988). The significance of dematin’s actin-bundling activity in erythrocytes is unknown. Actin bundles are not present in mature erythrocytes but do exist in early erythroblasts where dematin’s catalytic activity may be functionally significant (Koury et al. 1989). The primary sequence of dematin consists of an aminoterminal core domain of unknown function and a carboxy-terminal domain homologous to the “headpiece” domain of villin, an actinbinding protein of the brush border cytoskeleton (Rana et al. 1993; Arpin et al. 1988). This domain has since been identified in several other proteins including limatin (abLIM), supervillin, and advillin (Roof et al. 1997; Pestonjamasp et al. 1997; Marks et al. 1998). Transfection and mutagenesis studies of villin cDNAs revealed the headpiece domain to be crucial in microvilli morphogenesis (Friederich et al. 1992). While the normal development of microvilli in villin knockout mice seems to contradict the importance of villin, this observation suggests that dematin and/or other headpiececontaining proteins may compensate for villin function in the absorptive epithelia (Pinson et al. 1998). Here we report the cDNA sequence, genomic organization, and the mRNA expression of mouse dematin in fetal and adult tissues. The primary characterization of mouse erythroid dematin is the critical first step in generating knockout mice to study dematin function. We used RT-PCR and RACE analysis on cDNA reverse transcribed from mouse spleen mRNA to amplify overlapping fragments of the complete mouse dematin cDNA. The full-length cDNA sequence is∼2.3 kb in length, encoding an open reading frame of 383 amino acids (Fig. 1). This murine polypeptide corresponds to the 48-kDa isoform of human dematin. We also isolated transcripts encoding the 52-kDa polypeptide of dematin in which there is a 22-amino acid insertion (I-52) within the headpiece domain (Fig. 1B; Azim et al. 1995). We have previously demonstrated that this insertion, encoded by exon 13, binds ATP in vitro (Azim et al. 1996) and is homologous to a segment of protein 4.2 (Azim et al. 1995). The 5 8 noncoding sequence is 149 bp in length, while the 38 noncoding sequence is approximately 1.0 kb in length. A comparison of the 3 8 noncoding nucleotide sequence of mouse and human dematin cDNA indicates ∼60% sequence identity, suggesting a conservation of function across the two species. The coding sequence of mouse dematin displays 95% identity with the human sequence (Fig. 1C). Such striking similarity supports the notion that dematin knockout mice are likely to shed light on the physiological function of the human dematin gene. To elucidate the mouse dematin gene organization, we used a primer pair derived from the 3 8 noncoding region to screen a mouse P1 genomic library (Genome Systems Inc., St. Louis, MO). P1 clones #7227 and #7228 were determined to contain the entire dematin gene by Southern blot analysis (data not shown). Intronexon boundaries were determined by amplifying genomic fragments from the P1 clones with primers derived from the mouse dematin cDNA. PCR products were subcloned into pCR2.1 (Invitrogen Inc., Carlsbad, CA) for sequence analysis. The mouse dematin gene, in conservation with the human gene, is composed of 15 exons interrupted by 14 introns (Fig. 2A; Kim et al. 1998). All but one of the intron-exon boundaries, that between intron 7 and exon 8, conform to the eukaryotic 5 8 donor and 38 acceptor consensus splice junction sequence GT-AG (data reviewed but not shown). The positions of the intron-exon boundaries reveal the mouse exon sizes to be identical to that of human dematin (Kim et al. 1998). Exons 4 and 5 encode a PEST motif, while exon 8 contains a polyacidic motif. The headpiece domain of dematin is encoded by exons 10–15. Although the precise function of the PEST motif in dematin is not known, the proline-rich PEST signature sequence usually marks signaling proteins for regulated proteolysis in vivo (Rana et al. 1993). Similarly, the polyacidic motif in dematin may function in the subcellular targeting of the protein and in the formation of dematin-mediated multiprotein complexes (Rana et al. 1993). The approximate locations of the proline-rich motif (PEST), polyacidic motif, and the protein 4.2 homology motif in dematin are shown in Figure 2B. In addition, the carboxyl terminus of the headpiece domain of dematin contains a signature actin-binding motif that is found in a number of actinbinding proteins (Fig. 2B). Although originally isolated as a major component of the red cell cytoskeleton, dematin transcripts have been detected in a wide variety of tissues (Rana et al. 1993; Kim et al. 1998). To examine the expression pattern of mouse dematin, we performed Northern blot analysis of mRNA from fetal brain, spleen, liver, and reticulocytes. Fetal spleen and reticulocytes were obtained from phenylhydrazine-induced anemic mice. Interestingly, dematin expression in the fetal brain appears to be relatively low, contrary to the predominant message seen in adult brain (Fig. 3; Kim et al. 1998). Dematin is abundantly expressed in the fetal spleen and liver, the primary sites of erythropoiesis during embryogenesis, but it is Correspondence: A. Chishti at Biomedical Research ACH-404, St. Elizabeth’s Medical Center, 736 Cambridge Street, Boston, MA 02135, USA.

Exon skipping truncates the PDZ domain of human erythroid p55 in a patient with chronic myeloid leukemia in acute megakaryoblastic blast crisis

Human p55, the major palmitoylated protein associated with the cytoplasmic face of the erythrocyte membrane, is believed to modulate interactions between protein 4.1 and glycophorin C. It is the prototype of a newly described family of signaling molecules that includes hD1g, the human homologue of the Drosophila discs-large tumor suppressor protein. Chronic myeloid leukemia is characterized by transformation to a fulminating acute leukemia, heralded by evolution of the Philadelphia chromosome positive genotype (Ph +) to further abnormalities. RT-PCR of p55 mRNA from a patient with acute megakaryoblastic CML revealed a 69 base pair deletion in the PDZ domain, corresponding to exon 5 of the p55 gene. The deletion of constitutive exon 5 not only marks the first abnormality of the p55 cDNA in human disease but also the first abnormality of a PDZ domain in human disease and may represent another genetic abnormality associated with CML in blast crisis.

Localization of the gene encoding the erythrocyte membrane protein p55 (Mpp1) on the mouse X Chromosome.

Species: Mouse Locus name: cyclin E Locus symbol: Ccne Map position: D7Rp2-(1.9 +I. 1 cM)-Ccne-(4.3 + 1.6 cM)-[Lhb/ Klkl]. Lhb and Klkl have not been separated by recombination in this IB. DTRp2, Lhb, and Klkl have been mapped in this same interspecific backcross (IB) in previous studies [1-3]; information regarding these probes can be obtained from the original references. Method of mapping: Interspecific backcross (129/R1 x Mus spretus)Fj x 129/RI, N = 160 [2]. Molecular reagents: Mouse genomic DNA fragment sizes were determined by hybridization to Southern blots of a 32p-labeled 1808 base pair murine cyclin E cDNA insert [4]. Allele detection: lnterspecific hybrid and parental genomic DNAs were digested with Taql, electrophoresed, and blotted to Duralon membrane (Stratagene) for hybridization with random-primed radioactive probe as described [2]. The Mus spretus TaqI fragment is 3.6 kb, while the 129/RI Taql fragment is 5.1 kb. Published homologs: human [5], Xenopus [6], Drosophila [7]. Discussion: The human nuclear protein cyclin E is a regulatory subunit of the the cdc2-related protein kinase cdk2 and is required for the Gl-to-S transition during mitosis [5]. A murine bomolog with 75% sequence homology to the human cyclin E has been isolated from the mouse trophectodermal carcinoma cell line E6496B [4]. A Xenopus homolog appears to function in meiosis as well as mitosis ]61, and both it and a Drosophila [7] homolog appear to be developmentally regulated during embryogenesis. We have mapped the murine cyclin E by analysis of the segregation of a polymorphic Taql fragment in 160 progeny of a Mus spretus/Mus musculus interspecific backcross (IB) [2]. Comparison of the segregation pattern of Ccne with those of published loci previously mapped to this IB places Ccne between D7Rp2 and [Lhb/Klkl] on mouse Chromosome (Chr) 7 (Fig. 1). The human homolog, CCNE, has been mapped by fluores-