Faculty Bibliography

The small Ras-related GTP binding and hydrolyzing protein Ran has been implicated in a variety of processes, including cell cycle progression, DNA synthesis, RNA processing, and nuclear-cytosolic trafficking of both RNA and proteins. Like other small GTPases, Ran appears to function as a switch: Ran-GTP and Ran-GDP levels are regulated both by guanine nucleotide exchange factors and GTPase activating proteins, and Ran-GTP and Ran-GDP interact differentially with one or more effectors. One such putative effector, Ran-binding protein 1 (RanBP1), interacts selectively with Ran-GTP. Ran proteins contain a diagnostic short, acidic, carboxyl-terminal domain, DEDDDL, which, at least in the case of human Ran, is required for its role in cell cycle regulation. We show here that this domain is required for the interaction between Ran and RanBP1 but not for the interaction between Ran and a Ran guanine nucleotide exchange factor or between Ran and a Ran GTPase activating protein. In addition, Ran lacking this carboxyl-terminal domain functions normally in an in vitro nuclear protein import assay. We also show that RanBP1 interacts with the mammalian homolog of yeast protein RNA1, a protein involved in RNA transport and processing. These results are consistent with the hypothesis that Ran functions directly in at least two pathways, one, dependent on RanBP1, that affects cell cycle progression and RNA export, and another, independent of RanBP1, that affects nuclear protein import

Previous studies in the laboratory indicated that glycosylphosphatidylinositol (GPI)-anchored proteins may generate diversity of the cell surface of different neuronal populations (Rosen et al., 1992). In this study, we have extended these findings and surveyed the expression of GPI-anchored proteins in the developing rat CNS. In addition to several well characterized GPI-anchored cell adhesion molecules (CAMs), we detected an unidentified broad band of 65 kDa that is the earliest and most abundantly expressed GPI-anchored species in the rat CNS. Purification of this protein band revealed that it is comprised of several related proteins that define a novel subfamily of immunoglobulin-like (Ig) CAMs. One of these proteins is the opiate binding-cell adhesion molecule (OBCAM). We have isolated a cDNA encoding a second member of this family, that we have termed neurotrimin, and present evidence for the existence of additional family members. Like OBCAM, with which it shares extensive sequence identity, neurotrimin contains three immunoglobulin-like domains. Both proteins are encoded by distinct genes that may be clustered on the proximal end of mouse chromosome 9. Characterization of the expression of neurotrimin and OBCAM in the developing CNS by in situ hybridization reveals that these proteins are differentially expressed during development. Neurotrimin is expressed at high levels in several developing projection systems: in neurons of the thalamus, subplate, and lower cortical laminae in the forebrain and in the pontine nucleus, cerebellar granule cells, and Purkinje cells in the hindbrain. Neurotrimin is also expressed at high levels in the olfactory bulb, neural retina, dorsal root ganglia, spinal cord, and in a graded distribution in the basal ganglia and hippocampus. OBCAM has a much more restricted distribution, being expressed at high levels principally in the cortical plate and hippocampus. These results suggest that these proteins, together with other members of this family, provide diversity to the surfaces of different neuronal populations that could be important in the specification of neuronal connectivity

Ran genes encode a family of well-conserve small nuclear GTPases (Ras-related nuclear proteins), whose function is implicated in both normal cell cycle progression and the transport of RNA and proteins between the nucleus and the cytoplasm. Previous studies of Ran proteins have utilized cell-free systems, yeasts, and cultured mammalian cells. We have now characterized patterns of Ran gene expression in the mouse. Serum starvation suppressed Ran gene transcription in mouse 3T3 cells. Ran mRNA reappeared in cells within 3 h after refeeding. A single Ran mRNA species was detected at low levels in most somatic tissues of the adult mouse. In testis, this Ran mRNA was abundant, as were other larger transcripts. Analysis of testis-derived Ran cDNA clones revealed the presence of two transcripts, one specifying an amino acid sequence identical to that of human Ran/TC4 and one specifying an amino acid sequence 94% identical. Northern blotting and reverse transcriptase-PCR assays with oligonucleotide probes and primers specific for each transcript demonstrated that the isoform identical to Ran/TC4 was expressed in both somatic tissues and testis, while the variant form was transcribed only in testis. The existence of tissue-specific Ran isoforms may help to rationalize the diverse roles suggested for Ran by previous biochemical studies

Ran/TC4, a member of the RAS gene superfamily, encodes an abundant nuclear protein that binds and hydrolyzes GTP. Transient expression of a Ran/TC4 mutant protein deficient in GTP hydrolysis blocked DNA replication, suggesting a role for Ran/TC4 in the regulation of cell cycle progression. To test this possibility, we exploited an efficient transfection system, involving the introduction of cDNAs in the pMT2 vector into 293/Tag cells, to analyze phenotypes associated with mutant and wild-type Ran/TC4 expression. Expression of a Ran/TC4 mutant protein deficient in GTP hydrolysis inhibited proliferation of transfected cells by arresting them predominantly in the G2, but also in the G1, phase of the cell cycle. Deletion of an acidic carboxy-terminal hexapeptide from the Ran/TC4 mutant did not alter its nuclear localization but did block its inhibitory effect on cell cycle progression. These data suggest that normal progression of the cell cycle is coupled to the operation of a Ran/TC4 GTPase cycle. Mediators of this coupling are likely to include the nuclear regulator of chromosome condensation 1 protein and the mitosis-promoting factor complex

Although the human Ras proteins are members of a large superfamily of Ras-related proteins, to date, only the proteins encoded by the three mammalian ras genes have been found to possess oncogenic potential. Among the known Ras-related proteins, TC21/R-Ras2 exhibits the most significant amino acid identity (55%) to Ras proteins. We have generated mutant forms of TC21 that possess amino acid substitutions analogous to those that activate Ras oncogenic potential [designated TC21(22V) and TC21(71L)] and compared the biological properties of TC21 with those of Ras proteins in NIH 3T3 and Rat-1 transformation assays. Whereas wild-type TC21 did not show any transforming potential in vitro, both TC21(22V) and TC21(71L) displayed surprisingly potent transforming activities that were comparable to the strong transforming activity of oncogenic Ras proteins. Like Ras-transformed cells, NIH 3T3 cells expressing mutant TC21 proteins formed foci of morphologically transformed cells in monolayer cultures, proliferated in low serum, formed colonies in soft agar, and developed progressive tumors in nude mice. Thus, TC21 is the first Ras-related protein to exhibit potent transforming activity equivalent to that of Ras. Furthermore, mutant TC21 proteins also stimulated constitutive activation of mitogen-activated protein kinases as well as transcriptional activation from Ras-responsive promoter elements (Ets/AP-1 and NF-kappa B). We conclude that aberrant TC21 function may trigger cellular transformation via a signal transduction pathway similar to that of oncogenic Ras and suggest that deregulated TC21 activity may contribute significantly to human oncogenesis

The mouse congenital polycystic kidney (cpk) mutation causes bilateral cystic dilatation of the renal collecting tubules and leads to rapidly progressive renal insufficiency in affected homozygotes. The phenotype of the cpk/cpk mutants closely resembles that of human autosomal recessive polycystic kidney disease (ARPKD). Previously, we have reported that the cpk locus maps close to D12Nyu2 on Chromosome (Chr) 12. To determine the cpk map location more precisely, we have extended our previous studies using additional progeny and additional markers of proximal Chr 12. These recent studies position cpk within 1.3 cM of D12Nyu2, closely flanked by (Odc, D12Mit10) and (Tpo, D12Mit12). Our data support an ordered array of seven DNA markers that will provide reference points for building a physical map of the Chr 12 region centered on cpk. Moreover, these data establish that cpk lies within a linkage group that is conserved between mouse Chr 12 and human chr 2p24-2p25. This assignment to a region of homology will facilitate human linkage analyses to determine whether mouse cpk and human ARPKD are mutations of homologous genes

To refine the linkage map of mouse Chromosome (Chr) 12 and to define better the homology relationships between it and human chrs 2p and 14q, nine new anonymous DNA markers of Chr 12 were identified, and mouse loci homologous to the human D14S17, CHGA, HSPA2, RRM2, TPO, and ZFP50 ('KUP') genes were defined. The inheritance of DNA variants associated with these markers was followed in progeny of a reciprocal backcross between the C57BL/6J and SWR/J laboratory mouse strains and in recombinant inbred (RI) strains of mice. These data, combined with results of previous analyses of the backcross, allowed the construction of a 22-marker multilocus linkage map that spanned 58 cM. Use of this map to anchor the RI typing data collected in this and previous studies allowed the construction of a 79-marker map that spanned 66 cM and the identification of a framework of unambiguously ordered, extensively typed markers that should facilitate the use of RI mice in testing new markers for possible linkage to Chr 12

SH2 domain proteins are important components of the signal transduction pathways activated by growth factor receptor tyrosine kinases. We have been cloning SH2 domain proteins by bacterial expression cloning using the tyrosine phosphorylated C-terminus of the epidermal growth factor receptor as a probe. One of these newly cloned SH2 domain proteins, GRB-7, was mapped on mouse chromosome 11 to a region which also contains the tyrosine kinase receptor, HER2/erbB-2. The analogous chromosomal locus in man is often amplified in human breast cancer leading to overexpression of HER2. We find that GRB-7 is amplified in concert with HER2 in several breast cancer cell lines and that GRB-7 is overexpressed in both cell lines and breast tumors. GRB-7, through its SH2 domain, binds tightly to HER2 such that a large fraction of the tyrosine phosphorylated HER2 in SKBR-3 cells is bound to GRB-7. GRB-7 can also bind tyrosine phosphorylated SHC, albeit at a lower affinity than GRB2 binds SHC. We also find that GRB-7 has a strong similarity over > 300 amino acids to a newly identified gene in Caenorhabditis elegans. This region of similarity, which lies outside the SH2 domain, also contains a pleckstrin homology domain. The presence of evolutionarily conserved domains indicates that GRB-7 is likely to perform a basic signaling function. The fact that GRB-7 and HER2 are both overexpressed and bound tightly together suggests that this basic signaling pathway is greatly amplified in certain breast cancers