Putative Src Homology Research Articles

MOTS-c promotes muscle differentiation in vitro

MOTS-c (mitochondrial open reading frame of the 12 S rRNA-c) is a newly discovered peptide that has been shown to have a protective role in whole-body metabolic homeostasis. This could be a consequence of the effect of MOTS-c on muscle tissue. Here, we investigated the role of MOTS-c in the differentiation of human (LHCN-M2) and murine (C2C12) muscle progenitor cells. Cells were treated with peptides at the onset of differentiation or after myotubes had been formed. We identified in silico a putative Src Homology 2 (SH2) binding motif in the YIFY region of the MOTS-c sequence, and created a Y8F mutant MOTS-c peptide to explore the role of this region. In both cellular models, treatment with wild-type MOTS-c peptide increased myotube formation whereas treatment with the Y8F peptide did not. MOTS-c wild-type, but not Y8F peptide, also protected against interleukin-6 (IL-6)-induced reduction of nuclear myogenin staining in myocytes. Thus, we investigated whether MOTS-c interacts with the IL-6/Janus kinase/ Signal transducer and activator of transcription 3 (STAT3) pathway, and found that MOTS-c, but not the Y8F peptide, blocked the transcriptional activity of STAT3 induced by IL-6. Altogether, our findings suggest that, in muscle cells, MOTS-c interacts with STAT3 via the putative SH2 binding motif in the YIFY region to reduce STAT3 transcriptional activity, which enhances myotube formation. This newly discovered mechanism of action highlights MOTS-c as a potential therapeutic target against muscle-wasting in several diseases.

Fibroblast Growth Factor Receptor Like-1 (FGFRL1) Interacts with SHP-1 Phosphatase at Insulin Secretory Granules and Induces Beta-cell ERK1/2 Protein Activation

FGFRL1 is a newly identified member of the fibroblast growth factor receptor (FGFR) family expressed in adult pancreas. Unlike canonical FGFRs that initiate signaling via tyrosine kinase domains, the short intracellular sequence of FGFRL1 consists of a putative Src homology domain-2 (SH2)-binding motif adjacent to a histidine-rich C terminus. As a consequence of nonexistent kinase domains, FGFRL1 has been postulated to act as a decoy receptor to inhibit canonical FGFR ligand-induced signaling. In pancreatic islet beta-cells, canonical FGFR1 signaling affects metabolism and insulin processing. This study determined beta-cell expression of FGFRL1 as well as consequent effects on FGFR1 signaling and biological responses. We confirmed FGFRL1 expression at the plasma membrane and within distinct intracellular granules of both primary beta-cells and βTC3 cells. Fluorescent protein-tagged FGFRL1 (RL1) induced a significant ligand-independent increase in MAPK signaling. Removal of the histidine-rich domain (RL1-ΔHis) or entire intracellular sequence (RL1-ΔC) resulted in greater retention at the plasma membrane and significantly reduced ligand-independent ERK1/2 responses. The SHP-1 phosphatase was identified as an RL1-binding substrate. Point mutation of the SH2-binding motif reduced the ability of FGFRL1 to bind SHP-1 and activate ERK1/2 but did not affect receptor localization to insulin secretory granules. Finally, overexpression of RL1 increased cellular insulin content and matrix adhesion. Overall, these data suggest that FGFRL1 does not function as a decoy receptor in beta-cells, but rather it enhances ERK1/2 signaling through association of SHP-1 with the receptor's intracellular SH2-binding motif.

TRAF6 Protein Couples Toll-like Receptor 4 Signaling to Src Family Kinase Activation and Opening of Paracellular Pathway in Human Lung Microvascular Endothelia

Gram-negative bacteria release lipopolysaccharide (LPS) into the bloodstream. Here, it engages Toll-like receptor (TLR) 4 expressed in human lung microvascular endothelia (HMVEC-Ls) to open the paracellular pathway through Src family kinase (SFK) activation. The signaling molecules that couple TLR4 to the SFK-driven barrier disruption are unknown. In HMVEC-Ls, siRNA-induced silencing of TIRAP/Mal and overexpression of dominant-negative TIRAP/Mal each blocked LPS-induced SFK activation and increases in transendothelial [(14)C]albumin flux, implicating the MyD88-dependent pathway. LPS increased TRAF6 autoubiquitination and binding to IRAK1. Silencing of TRAF6, TRAF6-dominant-negative overexpression, or preincubation of HMVEC-Ls with a cell-permeable TRAF6 decoy peptide decreased both LPS-induced SFK activation and barrier disruption. LPS increased binding of both c-Src and Fyn to GST-TRAF6 but not to a GST-TRAF6 mutant in which the three prolines in the putative Src homology 3 domain-binding motif (amino acids 461-469) were substituted with alanines. A cell-permeable decoy peptide corresponding to the same proline-rich motif reduced SFK binding to WT GST-TRAF6 compared with the Pro → Ala-substituted peptide. Finally, LPS increased binding of activated Tyr(P)(416)-SFK to GST-TRAF6, and preincubation of HMVEC-Ls with SFK-selective tyrosine kinase inhibitors, PP2 and SU6656, diminished TRAF6 binding to c-Src and Fyn. During the TRAF6-SFK association, TRAF6 catalyzed Lys(63)-linked ubiquitination of c-Src and Fyn, whereas SFK activation increased tyrosine phosphorylation of TRAF6. The TRAF6 decoy peptide blocked both LPS-induced SFK ubiquitination and TRAF6 phosphorylation. Together, these data indicate that the proline-rich Src homology 3 domain-binding motif in TRAF6 interacts directly with activated SFKs to couple LPS engagement of TLR4 to SFK activation and loss of barrier integrity in HMVEC-Ls.

Functional Interaction of the Ras Effector RASSF5 with the Tyrosine Kinase Lck: Critical Role in Nucleocytoplasmic Transport and Cell Cycle Regulation

RASSF5 is a member of the Ras association domain family, which is known to be involved in cell growth regulation. Expression of RASSF5 is extinguished selectively by epigenetic mechanism(s) in different cancers and cell lines, and reexpression usually suppresses cell proliferation and tumorigenicity. To date, the mechanism regulating RASSF5 nuclear transport and its role in cell growth regulation remains unclear. Using heterokaryon assay, we have demonstrated that RASSF5 shuttles between the nucleus and the cytoplasm, and its export from the nucleus is sensitive to leptomycin B, suggesting that RASSF5 is exported from the nucleus by a CRM-1-dependent export pathway. We further demonstrate that RASSF5 contains a hydrophobic-rich nuclear export signal (NES) towards the C-terminus and two nuclear localization signals—one each at the N-terminus and the C-terminus. Combination of mutational and immunofluorescence analyses suggests that the functional NES residing between amino acids 260 and 300 in the C-terminus is necessary for the efficient export of RASSF5 from the nucleus. In addition, substitution of conserved hydrophobic residues within the minimal NES impaired RASSF5 export from the nucleus. Furthermore, exchange of proline residues within the putative Src homology 3 binding motifs altered the export of RASSF5 from the nucleus despite the presence of functional NES, suggesting that multiple domains independently modulate the nucleocytoplasmic transport of RASSF5. Interestingly, the present investigation provided evidence that RASSF5 interacts with the tyrosine kinase Lck through its C-terminal Src homology 2 binding motif and showed that Lck-mediated phosphorylation is critical for the efficient translocation of RASSF5 into the nuclear compartment. Interestingly, our data demonstrate that wild type and nuclear export defective (ΔNES) mutant of RASSF5 but not the import defective mutant of accumulate the cells at G1/S phase and induce apoptosis. Furthermore, the Lck-interaction-defective mutant of RASSF5 induces apoptosis without altering cell cycle progression, suggesting that RASSF5 induces apoptosis independent of cell cycle arrest. Together, our data demonstrate that interaction with Lck is critical for RASSF5 phosphorylation, which in turn regulates the cell growth control activity of RASSF5. Finally, we have shown that RASSF5 encodes four splice variants and is translocated to the nucleus by the classical nuclear import pathway. One of the splice variants, RASSF5C, was found to be localized in the cytoplasm and translocated into the nucleus upon leptomycin B treatment despite the absence of N-terminal nuclear localization signal, suggesting that distribution of RASSF5 variants in different cellular compartments may be critical for Ras-dependent cell growth regulation. Collectively, the present investigation provided evidence that Lck-mediated phosphorylation regulates the nucleocytoplasmic shuttling and cell growth control activities of RASSF5.

Isolation, Sequence Identification and Tissue Expression Distribution of Three Novel Porcine Genes-UBC1, TRA2A and SH21A

Liu G.Y., Zhao, S.M., Song, X.L., Wang, J. and Gao, S.Z. 2008. Isolation, sequence identification and tissue expression distribution of three novel porcine genes-UBC1, TRA2A and SH21A. J. Appl. Anim. Res., 33:33–38. Ubiquitin-conjugating enzyme E2–25 kDa (UBC1), transformer-2 protein homolog TRA-2 alpha (TRA2A) and SH2 domain protein 1A (SH21A) genes are most important genes highly related or potentially related with the porcine production. The complete coding sequences of these three porcine genes were amplified using the reverse transcriptase polymerase chain reaction (RT-PCR) based on the conserved sequence information of the mouse or other mammals and referred highly homologous pig ESTs. Porcine UBC1 gene encodes a protein of 200 amino acids which contains the conserved putative UBCc domain and has high homology with the UBC1 proteins of three species-human, mouse and bovine (100%). The porcine TRA2A gene encodes a protein of 279 amino acids that contains the conserved putative RNA recognition motif (RRM) domain and has high homology with the TRA2A proteins of two species-human (99%) and mouse (97%). The porcine SH21A gene encodes a protein of 128 amino acids that contains the conserved putative src homology 2 (SH2) domain and has high homology with the SH21A proteins of two species human (92%) and mouse (86%). The tissue expression analysis indicated that the swine UBC1, TRA2A and SH21A genes were expressed in different tissues including lung, fat, small intestine, large intestine, liver, muscle, spleen and kidney. Our experiment is the first to establish the primary foundation for further research on these three swine genes.

Functional characterization of the M-type K15-encoded membrane protein of Kaposi's sarcoma-associated herpesvirus

Kaposi's sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8 is the causative agent of Kaposi's sarcoma, primary effusion lymphoma and the plasma-cell variant of multicentric Castleman's disease. Its alternatively spliced K15 gene encodes several membrane proteins with varying numbers of transmembrane domains. Two highly diverged alleles of the K15 gene, termed predominant (P) and minor (M), exist and share only 33 % amino acid identity with one another, but retain conserved putative src homology (SH) 2- and SH3-binding motifs. K15-M is thought to have entered the KSHV genome as the result of recombination with a related gamma(2)-herpesvirus. The more common K15-P allele has been shown to activate the mitogen-activated protein kinases Erk2 and JNK1 and the nuclear factor kappaB (NF-kappaB) pathway. To explore possible functional differences between K15-P and K15-M that might have influenced their spread in the KSHV population, here, the ability of the M form of K15 to activate these pathways was investigated. Similarly to K15-P, K15-M induces the activation of the Erk2 and JNK1 kinases, the NF-kappaB transcription factor and the expression of a similar range of cellular inflammatory genes, as assessed by gene-expression microarray studies and reporter assays. In epithelial cells, the activation of most K15-M target genes is impaired by mutagenesis of Y(490) in its SH2-binding motif Y(490)EEV, although this motif appears less important in endothelial cells. Therefore, K15-M and K15-P can trigger similar intracellular signalling pathways, despite their extensive sequence divergence.

A Putative Src Homology 3 Domain Binding Motif but Not the C-terminal Dystrophin WW Domain Binding Motif Is Required for Dystroglycan Function in Cellular Polarity in Drosophila

The conserved dystroglycan-dystrophin (Dg.Dys) complex connects the extracellular matrix to the cytoskeleton. In humans as well as Drosophila, perturbation of this complex results in muscular dystrophies and brain malformations and in some cases cellular polarity defects. However, the regulation of the Dg.Dys complex is poorly understood in any cell type. We now find that in loss-of-function and overexpression studies more than half (34 residues) of the Dg proline-rich conserved C-terminal regions can be truncated without significantly compromising its function in regulating cellular polarity in Drosophila. Notably, the truncation eliminates the WW domain binding motif at the very C terminus of the protein thought to mediate interactions with dystrophin, suggesting that a second, internal WW binding motif can also mediate this interaction. We confirm this hypothesis by using a sensitive fluorescence polarization assay to show that both WW domain binding sites of Dg bind to Dys in humans (K(d) = 7.6 and 81 microM, respectively) and Drosophila (K(d) = 16 and 46 microM, respectively). In contrast to the large deletion mentioned above, a single proline to an alanine point mutation within a predicted Src homology 3 domain (SH3) binding site abolishes Dg function in cellular polarity. This suggests that an SH3-containing protein, which has yet to be identified, functionally interacts with Dg.

Structural Analysis of the Plakin Domain of Bullous Pemphigoid Antigen1 (BPAG1) Suggests that Plakins Are Members of the Spectrin Superfamily

Bullous pemphigoid antigen 1 (BPAG1) is a member of the plakin family of proteins. The plakins are multi-domain proteins that have been shown to interact with microtubules, actin filaments and intermediate filaments, as well as proteins found in cellular junctions. These interactions are mediated through different domains on the plakins. The interactions between plakins and components of specialized cell junctions such as desmosomes and hemidesmosomes are mediated through the so-called plakin domain, which is a common feature of the plakins. We report the crystal structure of a stable fragment from BPAG1, residues 226–448, defined by limited proteolysis of the whole plakin domain. The structure, determined by single-wavelength anomalous diffraction phasing from a selenomethionine-substituted crystal at 3.0 Å resolution, reveals a tandem pair of triple helical bundles closely related to spectrin repeats. Based on this structure and analysis of sequence conservation, we propose that the architecture of plakin domains is defined by two pairs of spectrin repeats interrupted by a putative Src-Homology 3 (SH3) domain.

EspF of Enteropathogenic Escherichia coli Binds Sorting Nexin 9

EspF of enteropathogenic Escherichia coli targets mitochondria and subverts a number of cellular functions. EspF consists of six putative Src homology 3 (SH3) domain binding motifs. In this study we identified sorting nexin 9 (SNX9) as a host cell EspF binding partner protein, which binds EspF via its amino-terminal SH3 region. Coimmunoprecipitation and confocal microscopy showed specific EspF-SNX9 interaction and non-mitochondrial protein colocalization in infected epithelial cells.

Atypical Mechanism of Regulation of the Wrch-1 Rho Family Small GTPase

Rho family GTPases are GDP/GTP-regulated molecular switches that regulate signaling pathways controlling diverse cellular processes. Wrch-1 was identified as a Wnt-1 regulated Cdc42 homolog, upregulated by Wnt1 signaling in Wnt1-transformed mouse mammary cells, and was able to promote formation of filopodia and activate the PAK serine/threonine kinase. Wrch-1 shares significant sequence and functional similarity with the Cdc42 small GTPase. However, Wrch-1 possesses a unique N-terminal 46 amino acid sequence extension that contains putative Src homology 3 (SH3) domain-interacting motifs. We determined the contribution of the N terminus to Wrch-1 regulation and activity. We observed that Wrch-1 possesses properties that distinguish it from Cdc42 and other Rho family GTPases. Unlike Cdc42, Wrch-1 possesses an extremely rapid, intrinsic guanine nucleotide exchange activity. Although the N terminus did not influence GTPase or GDP/GTP cycling activity in vitro, N-terminal truncation of Wrch-1 enhanced its ability to interact with and activate PAK and to cause growth transformation. The N terminus associated with the Grb2 SH3 domain-containing adaptor protein, and this association increased the levels of active Wrch-1 in cells. We propose that Grb2 overcomes N-terminal negative regulation to promote Wrch-1 effector interaction. Thus, Wrch-1 exhibits an atypical model of regulation not seen in other Rho family GTPases.

Tyrosine phosphorylation of QKI mediates developmental signals to regulate mRNA metabolism.

The selective RNA-binding protein QKI is essential for myelination in the central nervous system (CNS). QKI belongs to the family of signal transduction activators of RNA (STARs), characteristic of binding RNA and signaling molecules, therefore is postulated to regulate RNA homeostasis in response to developmental signals. Here we report that QKI acts downstream of the Src family protein tyrosine kinases (Src-PTKs) during CNS myelination. QKI selectively interacted with the mRNA encoding the myelin basic protein (MBP). Such interaction stabilized MBP mRNA and was required for the rapid accumulation of MBP mRNA during active myelinogenesis. We found that the interaction between QKI and MBP mRNA was negatively regulated by Src-PTK-dependent phosphorylation of QKI. During early myelin development, tyrosine phosphorylation of QKI in the developing myelin drastically declined, presumably leading to enhanced interactions between QKI and MBP mRNA, which was associated with the rapid accumulation of MBP mRNA and accelerated myelin production. Therefore, developmental regulation of Src-PTK-dependent tyrosine phosphorylation of QKI suggests a novel mechanism for accelerating CNS myelinogenesis via regulating mRNA metabolism.

Comparative analysis of the Band 4.1/ezrin-related protein tyrosine phosphatase Pez from two Drosophila species: implications for structure and function

The FERM-PTPs are a group of proteins that have FERM (Band 4.1, ezrin, radixin, moesin homology) domains at or near their N-termini, and PTP (protein tyrosine phosphatase) domains at their C-termini. Their central regions contain either PSD-95, Dlg, ZO-1 homology domains or putative Src homology 3 domain binding sites. The known FERM-PTPs fall into three distinct classes, which we name BAS, MEG, and PEZ, after representative human PTPs. Here we analyze Pez, a novel gene encoding the single PEZ-class protein present in Drosophila. Pez cDNAs were sequenced from the distantly related flies Drosophila melanogaster and Drosophila silvestris, and found to be highly conserved except in the central region, which contains at least 21 insertions and deletions. Comparison of fly and human Pez reveals several short conserved motifs in the central region that are likely protein binding sites and/or phosphorylation sites. We also identified novel invertebrate members of the BAS and MEG classes using genome data, and generated an alignment of vertebrate and invertebrate FERM domains of each class. ‘Specialized’ residues were identified that are conserved only within a given class of PTPs. These residues highlight surface regions that may bind class-specific ligands; for PEZ, these residues cluster on and near FERM subdomain F1. Finally, the PTP domain of fly Pez was modeled based on known PTP tertiary structures, and we conclude that Pez is likely a functional phosphatase despite some unusual features of the active site cleft sequences. Biochemical confirmation of this hypothesis and genetic analysis of Pez are currently underway.

Cloning and characterization of AASPs: novel axon-associated SH3 binding-like proteins.

Two cDNAs encoding closely related proteins were isolated from a crayfish nervous system lambdagtl0 cDNA library with a rat synapsin Ia cDNA probe. These proteins were expressed exclusively in neurons, were highly enriched in axons of the crayfish, and contained multiple, overlapping, putative Src homology 3 (SH3) binding sites. In concert with other proteins containing Src homology domains, SH3 binding proteins are thought to mediate protein-protein interactions in receptor signaling processes and with the cytoskeleton. We have named these proteins axon-associated SH3 binding-like proteins (AASPs). Except for these SH3 binding regions, which are also found in synapsins, AASPs were unlike any proteins in the database. AASPs were differentially expressed among motoneuron populations in crayfish and were found in growing axons and growth cones in culture. Affinity purified polyclonal antibodies to AASP-168 recognized immunoreactive proteins in rat and Xenopus, suggesting that AASPs may be conserved across species. Although the cellular function of AASPs is unclear at this time, they appear to be novel members of a neuron-specific SH3 binding protein family, which includes the synapsins.

Molecular cloning of a putative cyclic nucleotide-gated ion channel cDNA from Limulus polyphemus.

Cyclic nucleotide-gated channels have been proposed to mediate the electrical response to light in the ventral photoreceptor cells of the horseshoe crab, Limulus polyphemus. However, a cyclic nucleotide-gated channel has not been identified from Limulus. We have cloned a putative full-length cyclic nucleotide-gated channel cDNA by screening cDNA libraries constructed from Limulus brain using a probe developed from Limulus ventral eye nerves. The putative full-length cDNA was derived from two overlapping partial cDNA clones. The open reading frame encodes 905 amino acids; the sequence shows 44% identity to that of the alpha subunit of the bovine rod cyclic GMP-gated channel over the region containing the transmembrane domains and the cyclic nucleotide binding domain. This Limulus channel has a novel C-terminal region of approximately 200 amino acids, containing three putative Src homology domain 3 binding motifs and a putative coiled-coil domain. The possibility that this cloned channel is the same as that detected previously in excised patches from the photoreceptive membrane of Limulus ventral photoreceptors is discussed in terms of its sequence and its expression in the ventral eye nerves.

Dap160, a Neural-specific Eps15 Homology and Multiple SH3 Domain-containing Protein That Interacts with DrosophilaDynamin

The discovery of overlapping hot spots of dynamin (Estes, P. S., Roos, J., van der Bliek, A., Kelly, R. B., Krishnan, K. S., and Ramaswami, M. (1996) J. Neurosci. 16, 5443-5456) and the heterotetrameric adaptor 2 complex (Gonzalez-Gaitan, M., and Jäckle, H. (1997) Cell 88, 767-776) in Drosophila nerve terminals led to the concept of zones of active endocytosis close to sites of active exocytosis. The proline-rich domain of Drosophila dynamin was used to identify and purify a third component of the endocytosis zones. Dap160 (dynamin-associated protein 160 kDa) is a membrane-associated, dynamin-binding protein of 160 kDa that has four putative src homology 3 domains and an Eps15 homology domain, motifs frequently found in proteins associated with endocytosis. The binding capacities of the four putative src homology 3 domains were examined individually and in combination and shown to bind known proteins that contained proline-rich domains. Each binding site, however, was different in its preference for binding partners. We suggest that Dap160 is a scaffolding protein that helps anchor proteins required for endocytosis at sites where they are needed in the Drosophila nerve terminal.

Molecular cloning of the cDNA encoding pp36, a tyrosine-phosphorylated adaptor protein selectively expressed by T cells and natural killer cells.

Activation of T and natural killer (NK) cells leads to the tyrosine phosphorylation of pp36 and to its association with several signaling molecules, including phospholipase Cgamma-1 and Grb2. Microsequencing of peptides derived from purified rat pp36 protein led to the cloning, in rat and man, of cDNA encoding a T- and NK cell-specific protein with several putative Src homology 2 domain-binding motifs. A rabbit antiserum directed against a peptide sequence from the cloned rat molecule recognized tyrosine phosphorylated pp36 from pervanadate-treated rat thymocytes. When expressed in 293T human fibroblast cells and tyrosine-phosphorylated, pp36 associated with phospholipase Cgamma-1 and Grb2. Studies with GST-Grb2 fusion proteins demonstrated that the association was specific for the Src homology 2 domain of Grb-2. Molecular cloning of the gene encoding pp36 should facilitate studies examining the role of this adaptor protein in proximal signaling events during T and NK cell activation.

A novel gene that encodes a protein with a putative src homology 3 domain is a candidate gene for familial juvenile nephronophthisis.

Familial juvenile nephronophthisis (NPH) is an autosomal recessive, genetically heterogeneous disorder, representing the most frequent inherited cause of chronic renal failure in children. One of the responsible loci, NPH1 , has been mapped to 2q13. The presence of large homozygous deletions of approximately 250 kb in the majority of affected patients allowed us to define a minimal deletion interval for NPH1 . A BAC contig covering this interval was established. Combination of large scale genomic sequencing, cDNA selection and computer-aided analysis led to the characterization of two transcriptional units. One encodes the already known BENE protein, and the other encodes a novel protein of at least 732 amino acids containing a putative src homology 3 domain. In two patients carrying the large deletion of the NPH1 region on only one allele, two mutations were detected in two independent exons of the novel gene. One consists of a single base deletion, causing a frameshift, and the other is a G-->A substitution in the consensus 5' splice donor site. Both mutations thus potentially generate null mutants. One of these mutations was found to segregate with the disease in the family, and the second appeared to be a de novo mutation. We therefore conclude that this novel gene is a strong candidate for NPH.

Mutation analysis of the mouse myosin VIIA deafness gene.

The shaker-1 (Myo7a) mouse deafness locus is encoded by an unconventional myosin gene: myosin VIIA [Gibson, Walsh, Mburu, Varela, Brown, Antonio, Biesel, Steel and Brown (1995) Nature (London) 374, 62-64]. The myosin VIIA gene is expressed in hair cells in the cochlea, where it is thought to function in the development of the critical neuroepithelium where auditory transduction takes place. In order to understand better the function of myosin VIIA, we have determined the complete sequence of the mouse myosin VIIA cDNA and employed the wild-type sequence for mutational analysis of a number of shaker-1 alleles. Analysis of the mouse myosin VIIA tail sequence demonstrates a large internal repeat with regions of similarity to myosins IV, X and XII as well as members of the band 4.1 family. In addition, the myosin VIIA repeats are similar along their entire length to a tail domain from a plant kinesin. The mouse myosin VIIA tail also contains a putative Src homology 3 (SH3) domain. Along with three previously reported shaker-1 mutations, mutations for seven shaker-1 alleles in total have now been identified. The mutational changes have been analysed in terms of their predicted effect on both myosin motor head and tail domain function and the predictions related to the known phenotypes of the shaker-1 alleles. Five of the mutations lie in the motor head, and analysis of their likely effect on myosin head structure correlates well with the known severity of the shaker-1 alleles. Of the two mutations in the tail, one is a missense mutation within the kinesin and myosin IV, X and XII homology domains that substitutes a conserved amino acid and leads to a severe deafness phenotype. This and other data suggest that myosin VIIA may have properties of a myosin-motor-kinesin-tail hybrid and be involved in membrane turnover within the actin-rich environment of the apical hair cell surface.

The EH-domain-containing protein Pan1 is required for normal organization of the actin cytoskeleton in Saccharomyces cerevisiae.

Normal cell growth and division in the yeast Saccharomyces cerevisiae involve dramatic and frequent changes in the organization of the actin cytoskeleton. Previous studies have suggested that the reorganization of the actin cytoskeleton in accordance with cell cycle progression is controlled, directly or indirectly, by the cyclin-dependent kinase Cdc28. Here we report that by isolating rapid-death mutants in the background of the Start-deficient cdc28-4 mutation, the essential yeast gene PAN1, previously thought to encode the yeast poly(A) nuclease, is identified as a new factor required for normal organization of the actin cytoskeleton. We show that at restrictive temperature, the pan1 mutant exhibited abnormal bud growth, failed to maintain a proper distribution of the actin cytoskeleton, was unable to reorganize actin the cytoskeleton during cell cycle, and was defective in cytokinesis. The mutant also displayed a random pattern of budding even at permissive temperature. Ectopic expression of PAN1 by the GAL promoter caused abnormal distribution of the actin cytoskeleton when a single-copy vector was used. Immunofluorescence staining revealed that the Pan1 protein colocalized with the cortical actin patches, suggesting that it may be a filamentous actin-binding protein. The Pan1 protein contains an EF-hand calcium-binding domain, a putative Src homology 3 (SH3)-binding domain, a region similar to the actin cytoskeleton assembly control protein Sla1, and two repeats of a newly identified protein motif known as the EH domain. These findings suggest that Pan1, recently recognized as not responsible for the poly(A) nuclease activity (A. B. Sachs and J. A. Deardorff, erratum, Cell 83:1059, 1995; R. Boeck, S. Tarun, Jr., M. Rieger, J. A. Deardorff, S. Muller-Auer, and A. B. Sachs, J. Biol. Chem. 271:432-438, 1996), plays an important role in the organization of the actin cytoskeleton in S. cerevisiae.

Molecular Cloning of the Human Homolog of a Striatum-Enriched Phosphatase (STEP) Gene and Chromosomal Mapping of the Human and Murine Loci

A gene for a protein tyrosine phosphatase (PTPase) was isolated from a human fetal brain cDNA library by PCR amplification. Sequence analysis revealed that the PTPase has a single phosphatase catalytic domain located at the C-terminus that includes the highly conserved amino acid domain [I/V]HCXAGXXR[S/T]GX[F/Y] found in all tyrosine phosphatases. Two proline-rich regions located at the N-terminus may contain putative Src homology domain 3 (SII3) binding motifs. Comparison of the PTPase with a previously cloned striatum enriched phosphatase (STEP) from rat and from mouse exhibited a high degree of identity (∼8590%) at both the nucleotide and the amino acid levels, indicating that the human PTPase is the homolog of the rat and murine STEP gene. By using a combination of somatic cell hybrid analysis and fluorescence in situ hybridization, we have mapped the human STEP locus to chromosome 11p15.2-p15.1 and the murine STEP gene to chromosome 7B3-B5. These are two regions of known conserved synteny, providing further evidence that the human STEP is a true homolog of the murine STEP gene. Candidate disease genes in the vicinity include Usher syndrome type IC in human and a mouse mutant locus, twister (twt).