Disruption of Abi1/Hssh3bp1 expression induces prostatic intraepithelial neoplasia in the conditional Abi1/Hssh3bp1 KO mice.

Prostate cancer is one of the leading causes of cancer-related deaths in the United States and a leading diagnosed non-skin cancer in American men. Genetic mutations underlying prostate tumorigenesis include alterations of tumor suppressor genes. We tested the tumor suppressor hypothesis for ABI1/hSSH3BP1 by searching for gene mutations in primary prostate tumors from patients, and by analyzing the consequences of prostate-specific disruption of the mouse Abi1/Hssh3bp1 ortholog. We sequenced the ABI1/hSSH3BP1 gene and identified recurring mutations in 6 out of 35 prostate tumors. Moreover, complementation and anchorage-independent growth, proliferation, cellular adhesion and xenograft assays using the LNCaP cell line, which contains a loss-of-function Abi1 mutation, and a stably expressed wild-type or mutated ABI gene, were consistent with the tumor suppressor hypothesis. To test the hypothesis further, we disrupted the gene in the mouse prostate by breeding the Abi1 floxed strain with the probasin promoter-driven Cre recombinase strain. Histopathological evaluation of mice indicated development of prostatic intraepithelial neoplasia (PIN) in Abi1/Hssh3bp1 knockout mouse as early as the eighth month, but no progression beyond PIN was observed in mice as old as 12 months. Observed decreased levels of E-cadherin, ß-catenin and WAVE2 in mouse prostate suggest abnormal cellular adhesion as the mechanism underlying PIN development owing to Abi1 disruption. Analysis of syngeneic cell lines point to the possibility that upregulation of phospho-Akt underlies the enhanced cellular proliferation phenotype of cells lacking Abi1. This study provides proof-of-concept for the hypothesis that Abi1 downregulation has a role in the development of prostate cancer.

Loss of expression of human spectrin src homology domain binding protein 1 is associated with 10p loss in human prostatic adenocarcinoma.

The gene encoding human spectrin Src homology domain binding protein 1, or Hssh3bp1, which is a marker of macropinocytic vesicles and a potential regulator of macropinocytosis, co-localizes to a YAC containing chromosome 10p sequences at loci D10S89 and D10S111 that are frequently deleted in prostate tumors. Expression of Hssh3bp1 was evaluated at the protein level in 17 paired normal and malignant prostate tumor samples using the monoclonal antibody 2G8 to Hssh3bp1. These experiments demonstrated that 4/6 tumors (67%) with 10p deletion failed to express Hssh3bp1 protein compared to 5/11 (46%) tumors with intact 10p. Thus, loss of Hssh3bp1 expression is concordant with allelic loss of adjacent 10p sequences in human prostate tumors. In addition, two prostate tumor cell lines contain an exon skipping mutation in the Hssh3bp1 gene that leads to the abnormal splicing of the mRNA and loss of a portion of Abl tyrosine kinase SH3 domain binding site in the protein. These data are consistent with a role for Hssh3bp1 as a candidate tumor suppressor gene inactivated during prostate tumorigenesis.

Monoclonal antibodies to alphaI spectrin Src homology 3 domain associate with macropinocytic vesicles in nonerythroid cells.

Spectrins represent a family of membrane-associated proteins responsible for membrane flexibility and cell shape in erythrocytes, and probably in most nonerythroid cells. Spectrin functions as a tetramer consisting of two heterodimers each containing two subunits termed alpha and beta. In humans, alphaI and alphaII spectrins but not beta spectrins are characterized by the presence of an Src homology 3 (SH3) domain. As a tool to investigate the function of spectrin SH3 domains we derived several monoclonal antibodies (mAb) to the recombinant human alphaI or alphaII spectrin SH3 domain. Immunostaining using these monoclonal antibodies indicated expression of alphaI spectrin in cell bodies and alphaII spectrin in neurites of granule neurons in mouse primary cerebellar cultures. Monoclonal antibodies reactive to alphaI spectrin SH3 domain indicated expression of a protein(s) containing an alphaI-like SH3 domain in cytoplasmic vesicular-like structures in GFAP-positive cells in these cultures. In NIH 3T3 fibroblasts, these antibodies label macropinocytic vesicles. Together, these data and Western blotting results suggest expression of at least three spectrin-SH3 domain antibody-reactive proteins.

Human spectrin Src homology 3 domain binding protein 1 regulates macropinocytosis in NIH 3T3 cells.

Macropinocytosis is an endocytic process that occurs through non-clathrin coated vesicles larger than 0.2 microm in diameter. Although macropinocytic vesicles are readily visualized in cultured cells by the introduction of fluorescent, water-soluble dyes into the culture medium, protein markers associated with this type of vesicles have not yet been well defined. Here, we report that human spectrin SH3 domain binding protein 1, or Hssh3bp1, associates with macropinosomes in NIH 3T3 fibroblasts. Hssh3bp1 macropinosomes are heterogeneous in morphology and size, do not endocytose transferrin and are resistant to brefeldin A treatment. Cytochalasin D, and wortmannin block endocytosis of fluorescent dyes into the Hssh3bp1 macropinosomes and dramatically affect their morphology. Overexpression of Hssh3bp1-green fluorescent protein abolished fusion of vesicles resulting in a decreased endocytosis of fluorescence dyes, thus suggesting a potential regulatory role of Hssh3bp1 in macropinocytosis. In the macropinosomes of NIH 3T3 cells, Hssh3bp1 associates with a 200-kDa protein that crossreacts with a monoclonal antibody to the erythroid alpha-spectrin SH3 domain. Thus macropinosomes in cells may contain a spectrin-like protein.

Two distinct mechanisms for regulation of nonmuscle myosin assembly via the heavy chain: phosphorylation for MIIB and mts 1 binding for MIIA.

In search of the regulation mechanisms for isoform specific myosin assembly, we have used the COOH-terminal fragments of nonmuscle myosin isoforms MIIA and MIIB (MIIA(F46) and MIIB(alpha)(F47)) as a model system. Phosphorylation by protein kinase C (PK C) or casein kinase II (CK II) within or near the nonhelical tail-end domain inhibits assembly of MIIB(alpha)(F47) [Murakami, N., et al. (1998) Biochemistry 37, 1989]. In the study presented here, we mutated the kinase sites to analyze the inhibition mechanisms of MIIB assembly by phosphorylation. Replacement of the CK II or PK C sites with Asp (MIIB(alpha)(F47)-CK-5D or -PK-4D) strongly inhibited the filament assembly, with or without Mg(2+), by significantly increasing the critical concentrations for assembly. Without Mg(2+), MIIB(alpha)(F47)-CK-5D or -PK-4D inhibited the assembly of wild-type (wt) MIIB(alpha)(F47) by either mixing as homofragments or forming heterofragments. With 2.5 mM Mg(2+), MIIB(alpha)(F47)-wt promoted assembly of MIIB(alpha)(F47)-CK-5D and -PK-4D in homofragment mixtures, but not by forming heterofragments. MIIA(F46) coassembled with MIIB(alpha)(F47)-wt and -CK-5D and altered their assembly patterns. In contrast, assembly of MIIB(alpha)(F47)-PK-4D was unchanged by MIIA(F46). A metastasis-associated protein, mts 1, bound in a Ca(2+)-dependent manner to MIIA(F46), but not appreciably to MIIB(alpha)(F47). At 0.15 M NaCl, mts 1-Ca(2+) not only inhibited MIIA(F46) assembly but also disassembled the MIIA(F46) filaments. Mts 1, however, did not affect the assembly of MIIB(alpha)(F47) in MIIA(F46) and MIIB(alpha)(F47) mixtures, indicating that mts 1 is an inhibitor specific to MIIA assembly. Our results suggest strongly that assembly of MIIA and MIIB is regulated by distinct mechanisms via tail-end domains: phosphorylation of MIIB and mts 1 binding to MIIA. These mechanisms may also function to form MIIA or MIIB homofilaments by selectively inhibiting MIIB or MIIA assembly.

Altered binding of mutated presenilin with cytoskeleton-interacting proteins.

The majority of familial Alzheimer's disease (AD) cases are linked to mutations on presenilin 1 and 2 genes (PS1 and PS2). The normal function of the proteins and the mechanisms underlying early-onset AD are currently unknown. To address this, we screened an expression library for proteins that bind differentially to the wild-type PS1 and mutant in the large cytoplasmic loop (PS1L). Thus we isolated the C-terminal tail of the 170 kDa cytoplasmic linker protein (CLIP-170) and Reed-Sternberg cells of Hodgkin's disease-expressed intermediate filament-associated protein (Restin), cytoplasmic proteins linking vesicles to the cytoskeleton. PS1L binding to CLIP-170/restin requires Ca(2+). Treating cells with thapsigargin or ionomycin increased the mutated PS1 in CLIP-170 immunoprecipitates. Further, PS1 and CLIP-170 co-localize in transfected cells and neuronal cultures.

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 (alphaI) SH3 domain as a bait. Five isoforms of the alphaI SH3 domain-binding protein mRNA were identified in human brain. Mapping of SH3 binding regions revealed the presence of two alphaI 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 alphaI SigmaI/betaI SigmaI antibody in untransfected NIH3T3 cells; in addition, the anti-alphaI SigmaI/betaI SigmaI antibody also stained Golgi apparatus. Immunofluorescence obtained using antibodies against alphaI SigmaI/++betaI SigmaI spectrin and Abl tyrosine kinase but not against alphaII/betaII 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.

Solution conformations of a peptide containing the cytoplasmic domain sequence of the beta amyloid precursor protein.

The cytoplasmic domain of the beta amyloid precursor protein (betaAPP) may play a role in cellular events that lead to the secretion of the Abeta peptide, the major constituent of amyloid plaques found in the brains of individuals affected by Alzheimer's disease, by interacting with cellular factors involved in betaAPP function or processing. In order to elucidate the structural basis of cytoplasmic domain activity, the conformations adopted in solution by a peptide containing the 47-residue C-terminal sequence of betaAPP have been investigated by NMR and CD spectroscopy. The peptide does not have a stable tertiary structure, but local regions of the polypeptide chain populate defined conformations. In particular, the amino acid sequences TPEE and NPTY form type I reverse turns. These structured regions correspond to sequences within the cytoplasmic domain implicated in the biological activity of betaAPP.

Physical properties of a single-motif erythrocyte spectrin peptide: a highly stable independently folding unit.

Spectrin is a long flexible rod-like actin cross-linking protein mostly comprised of many tandem homologous 106-residue motifs. In this study, the conformational stability and physical properties of a single homologous motif peptide, alpha1, were evaluated and compared to intact spectrin monomers and alphabeta heterodimers. It is interesting that while spectrin dimers elongate by about 3-fold in low ionic strength buffers relative to their size in physiological buffers, the single-motif peptide does not show significant changes in secondary structure in 10 mM phosphate buffer compared with isotonic buffer. This single-motif peptide is monomeric in physiological buffer as demonstrated by equilibrium sedimentation studies, and its hydrodynamic radius determined by gel filtration and dynamic light scattering of about 2.2 nm is consistent with an elongated rod-like shape. Unfolding of the single-motif peptide in urea solutions was similar to unfolding of intact heterodimers. Differential scanning calorimetry analyses showed that this single motif undergoes a reversible two-state transition with a Tm of 53 degrees C and an enthalpy of 65 kcal/mol in physiological buffer. Thermal stability was unaffected by ionic strength changes, but was decreased below physiological pH. These data show that this 13 kDa spectrin motif is a monomeric, highly stable, triple-helical, independently folding protein building block with physical characteristics that define many of the structural properties of the 526 kDa spectrin heterodimer. In contrast, interactions between adjacent motifs are probably responsible for spectrin's molecular flexibility and elasticity.

Molecular basis and haplotyping of the alphaII domain polymorphisms of spectrin: application to the study of hereditary elliptocytosis and pyropoikilocytosis.

Hereditary elliptocytosis (HE) and hereditary pyropoikilocytosis (HPP) are inherited disorders of erythrocyte shape that are frequently associated with abnormalities in alpha-spectrin, one of the principal structural proteins of the erythrocyte membrane skeleton. Five polymorphisms of the alpha-spectrin gene, located in a 6-kb interval of genomic DNA, were identified and analyzed in normal and mutant alpha-spectrin alleles. Three of these polymorphisms are due to single nucleotide substitutions in the alpha-spectrin gene coding region that lead to changes in the amino acid sequence. In combination, these three polymorphisms are responsible for the different peptide phenotypes of the alphaII domain previously observed following limited tryptic digestion of spectrin protein. The most common haplotype, type 1, was found predominantly in Caucasians and was the only haplotype identified in Asians. Haplotypes 2, 3, and 4 were identified predominantly in individuals of African ancestry and were commonly found in patients with HE or HPP. Analysis of coinheritance of alphaII domain polymorphisms with alpha-spectrin gene mutations causing HE or HPP in African-American patients with HE and HPP suggests that, with one exception, a given HE/HPP mutation is present in an alpha-spectrin gene of only one haplotype, indicating a founder effect. The other two polymorphisms located in this region of the alpha-spectrin gene do not change the amino acid sequence of the encoded alpha-spectrin chain and are not in linkage disequilibrium with three of the four alphaII domain haplotypes. A model is proposed for the evolutionary origin of the different haplotypes.

Mapping the human erythrocyte beta-spectrin dimer initiation site using recombinant peptides and correlation of its phasing with the alpha-actinin dimer site.

Human erythroid spectrin dimer assembly is initiated by the association of a specific region near the N-terminal of beta-spectrin with a complementary region near the C-terminal of alpha-spectrin (Speicher, D. W., Weglarz, L., and DeSilva, T. M. (1992) J. Biol. Chem. 267, 14775-14782). Both spectrin subunits consist primarily of tandem, 106-residue long, homologous, triple-helical motifs. In this study, the minimal region of beta-spectrin required for association with alpha-spectrin was determined using recombinant peptides. The start site (phasing) for construction of dimerization competent beta-spectrin peptides was particularly critical. The beginning of the first homologous motif for both beta-spectrin and the related dimerization site of alpha-actinin is approximately 8 residues earlier than most spectrin motifs. A four-motif beta-spectrin peptide (beta1-4+) with this earlier starting point bound to full-length alpha-spectrin with a Kd of about 10 nM, while deletion of these first 8 residues reduced binding nearly 10-fold. N- and C-terminal truncations of one or more motifs from beta1-4+ showed that the first motif was essential for dimerization since its deletion abolished binding, but beta1+ alone could not associate with alpha-monomers. The first two motifs (beta1 2+) represented the minimum lateral dimer assembly site with a Kd of about 230 nM for interaction with full-length alpha-spectrin or an alpha-spectrin nucleation site recombinant peptide, alpha18-21. Each additional motif increased the dimerization affinity by approximately 5-fold. In addition to this strong inter-subunit dimer association, interactions between the helices of a single triple-helical motif are frequently strong enough to maintain a noncovalent complex after internal protease cleavage similar to the interactions thought to be involved in tetramer formation. Analysis of hydrodynamic radii of recombinant peptides containing differing numbers of motifs showed that a single motif had a Stokes radius of 2.35 nM, while each additional motif added only 0.85 nM to the Stokes radius. This is the first direct demonstration that spectrin's flexibility arises from regions between each triple helical motif rather than from within the segment itself and suggests that current models of inter-motif connections may need to be revised.

Review. David Oppenheimer Memorial Lecture 1995: Some neuropathological aspects of Alzheimer's disease and its relevance to other disciplines.

Recent studies of diffuse A beta plaques point to the neurons as a source of A beta in diffuse plaques. The neuritic (primitive and classical) plaques appear to be the product of microglia and the myocytes are the source of amyloid deposits in the meningeal and cortical vessels. Dyshoric angiopathy is associated with deposits of amyloid by perivascular cells. Fibrillization of the neuron-derived diffuse, thioflavine-negative or benign plaques is poor or undetectable by current morphological methods including ultrastructural immunocytochemistry. It appears that fibrillization depends on the length of the A beta peptides and on the presence of amyloid-associated proteins. Four genes are now tightly linked with Alzheimer's disease (AD) and they are located on chromosomes 21, 19, 14 and 1. Therefore, AD should be considered a polyaetiological disease or syndrome. There are currently five transgenic mouse models overexpressing beta-APP. There is also a myocyte tissue culture model in which both soluble and fibrillized A beta are found. The relationship between A beta and neurofibrillary pathology is not clear and the current cascade hypothesis proposing that A beta pathology drives the formulation of neurofibrillary tangles is being questioned. There is growing evidence that it is not the A beta hypothesis, but the co-existing A beta neurofibrillary tangle pathology hypothesis which will be the basis for AD neuropathology.

Functional characterization of recombinant human red cell alpha-spectrin polypeptides containing the tetramer binding site.

Spectrin, a heterodimer composed of alpha and beta subunits, interacts with itself head-to-head to form tetramers in the erythrocyte membrane cytoskeleton. The NH2-terminal region of alpha-spectrin, encompassing the alpha I 80-kDa domain, was expressed in Escherichia coli. In addition to the correctly initiated polypeptide, four smaller polypeptides were produced by initiation at internal codons. Only the full-length polypeptide was able to bind to spectrin dimers, beta monomers, and to a recombinant polypeptide containing the COOH terminus of beta-spectrin. The head-to-head interaction with beta-spectrin was also retained by a recombinant polypeptide containing the NH2-terminal 158 amino acids of the alpha subunit. Deletion of the first 27 or 49 NH2-terminal amino acids abolished binding of this polypeptide to the beta monomer. The phasing used to design these recombinant polypeptides was based on a conformational model recently refined by Speicher et al. (Speicher, D. W., DeSilva, T. M., Speicher, K. D., Ursitti, J. A., Hembach, P., and Weglarz, L. (1993) J. Biol. Chem. 268, 4227-4235), where the structural unit begins and terminates around residue 30 of the repeat unit. The binding properties, mobility on gel filtration, and circular dichroism data of the recombinant polypeptides indicated that most polypeptides were able to assume their native conformation.

Low expression allele alpha LELY of red cell spectrin is associated with mutations in exon 40 (alpha V/41 polymorphism) and intron 45 and with partial skipping of exon 46.

The alpha V/41 polymorphism of erythroid alpha-spectrin has been characterized initially by an increased susceptibility to proteolysis of the alpha IV-alpha V domain junction (Alloisio N., L. Morlé, J. Maréchal, A.-F. Roux, M.-T. Ducluzeau, D. Guetarni, B. Pothier, F. Baklouti, A. Ghanem, R. Kastally, et al. 1991. J. Clin. Invest. 87:2169-2177). Until now, it has been found associated invariably with a low expression level of the corresponding alpha chain. Among 61 chromosomes investigated in French and North African individuals or kindreds, we observed 19 chromosomes with the alpha V/41 polymorphism. With no single exception, the latter displayed a point mutation in exon 40 (Leu-->Val; CTA-->GTA) at position alpha 1857. According to the triple helical model of spectrin structure, this change accounts for the peptide maps' abnormalities. Sequencing the entire alpha V domain cDNA disclosed, in addition, a partial skipping of exon 46. At the gene level, a substitution (C-->T) was evidenced at nucleotide -12 of intron 45. This mutation appeared linked to the exon 40 mutation in 17 chromosomes, again with no single exception, among 53 examined chromosomes. We hypothesized that the lack of exon 46 would hamper the nucleation process and eventually account for the low expression feature. The present doubly mutated allele was renamed allele alpha LELY (low expression, Lyon).

Evaluation of foreign gene codon optimization in yeast: expression of a mouse IG kappa chain.

We have optimized the codons in an immunoglobulin kappa chain gene to those preferred in the yeast Saccharomyces cerevisiae. The mutant and wild type kappa chain genes were each fused with a synthetic invertase signal peptide that also contained only yeast-preferred codons, and expressed in the F762 yeast strain. The use of yeast-preferred codons resulted in a more than 5-fold increase in the rate of synthesis and at least a 50-fold increase in the steady state level of protein.

The exon-intron organization of the human erythrocyte alpha-spectrin gene.

The human erythrocyte alpha-spectrin gene which spans 80 kbp has been cloned from human genomic DNA as overlapping lambda 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 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.

The complete cDNA and polypeptide sequences of human erythroid alpha-spectrin.

Overlapping human erythroid alpha-spectrin cDNA clones were isolated from lambda gt11 libraries constructed from cDNAs of human fetal liver and erythroid bone marrow. The composite 8001-base pair (bp) cDNA nucleotide sequence contains 187-bp 5'- and 528-bp 3'-untranslated regions and has a single long open reading frame of 7287 bp that encodes a polypeptide of 2429 residues. As previously described (Speicher, D. W., and Marchesi, V. T. (1984) Nature 311, 177-180), spectrin is composed largely of homologous 106-amino acid repeat units. From the amino acid sequence deduced from the cDNA, alpha-spectrin can be divided into 22 segments. Segments 1-9 and 12-19 are homologous and can therefore be considered repeats; the average number of identical residues in pairwise comparisons of these repeats is 22 out of 106, or 21%. Of these 17 repeats, 11 are exactly 106 amino acids in length, whereas five others differ from this length by a single residue. Segments 11, 20, and 21, although less homologous, appear to be related to the more highly conserved repeat units. The very N-terminal 22 residues, segment 10, which is atypical both in length and sequence, and the C-terminal 150 residues in segment 22 appear to be unrelated to the conserved repeat units. The sequence of the erythroid alpha-spectrin polypeptide chain is compared to that of human alpha-fodrin and chicken alpha-actinin to which it is related. alpha-Spectrin is more distantly related to dystrophin.