Leszek Kotula

Identification of a Candidate Human Spectrin Src Homology 3 Domain-binding Protein Suggests a General Mechanism of Association of Tyrosine Kinases with the Spectrin-based Membrane Skeleton

Spectrin is a widely expressed protein with specific isoforms found in erythroid and nonerythroid cells. Spectrin contains an Src homology 3 (SH3) domain of unknown function. A cDNA encoding a candidate spectrin SH3 domain-binding protein was identified by interaction screening of a human brain expression library using the human erythroid spectrin (αI) SH3 domain as a bait. Five isoforms of the αI SH3 domain-binding protein mRNA were identified in human brain. Mapping of SH3 binding regions revealed the presence of two αI SH3 domain binding regions and one Abl-SH3 domain binding region. The gene encoding the candidate spectrin SH3 domain-binding protein has been located to human chromosome 10p11.2 → p12. The gene belongs to a recently identified family of tyrosine kinase-binding proteins, and one of its isoforms is identical to e3B1, an eps8-binding protein (Biesova, Z., Piccoli, C., and Wong, W. T. (1997)Oncogene 14, 233–241). Overexpression of the green fluorescent protein fusion of the SH3 domain-binding protein in NIH3T3 cells resulted in cytoplasmic punctate fluorescence characteristic of the reticulovesicular system. This fluorescence pattern was similar to that obtained with the anti-human erythroid spectrin αIΣI/βIΣI antibody in untransfected NIH3T3 cells; in addition, the anti-αIΣI/βIΣI antibody also stained Golgi apparatus. Immunofluorescence obtained using antibodies against αIΣI/βIΣI spectrin and Abl tyrosine kinase but not against αII/βII spectrin colocalized with the overexpressed green fluorescent protein-SH3-binding protein. Based on the conservation of the spectrin SH3 binding site within members of this protein family and published interactions, a general mechanism of interactions of tyrosine kinases with the spectrin-based membrane skeleton is proposed.

The exon-intron organization of the human erythrocyte α-spectrin gene

Abstract The human erythrocyte α-spectrin gene which spans 80 kbp has been cloned from human genomic DNA as overlaping λ recombinants. The exon-intron junctions were identified and the exons mapped. The gene is encoded by 52 exons whose sizes range from 684 bp to the smallest of 18 bp. The donor and acceptor splice site sequences match the splice site consensus sequences, with the exception of one splice site where a donor sequence begins with -GC. The size and location of exons do not correlate with the 106-amino-acid repeat, except in three locations where the surrounding codons are conserved as well. The lack of correspondence between exons and the 106-amino-acid repeat is interpreted to reflect the appearance of a spectrin-like gene from a minigene early in the evolution of eukaryotes. Since current evidence indicates that introns were present in genes before the divergence of prokaryotes and eukaryotes, it is possible that the original distribution of introns within the minigene has been lost by the random deletion of introns from the spectrin gene.

Interaction of HIV-1 gp41 Core with NPF Motif in Epsin IMPLICATION IN ENDOCYTOSIS OF HIV

The human immunodeficiency virus, type 1 (HIV-1), gp41 core plays an important role in fusion between viral and target cell membranes. We previously identified an HIV-1 gp41 core-binding motif HXXNPF (where X is any amino acid residue). In this study, we found that Asn, Pro, and Phe were the key residues for gp41 core binding. There are two NPF motifs in Epsin-1-(470–499), a fragment of Epsin, which is an essential accessory factor of endocytosis that can dock to the plasma membrane by interacting with the lipid. Epsin-1-(470–499) bound significantly to the gp41 core formed by the polypeptide N36(L8)C34 and interacted with the recombinant soluble gp41 containing the core structure. A synthetic peptide containing the Epsin-1-(470–499) sequence could effectively block entry of HIV-1 virions into SupT1 T cells via the endocytosis pathway. These results suggest that interaction between Epsin and the gp41 core, which may be present in the target cell membrane, is probably essential for endocytosis of HIV-1, an alternative pathway of HIV-1 entry into the target cell.