We have used mouse embryonic fibroblasts (MEFs) devoid of Ras proteins to illustrate that they are essential for proliferation and migration, but not for survival, at least in these cells. These properties are unique to the Ras subfamily of proteins because ectopic expression of other Ras-like small GTPases, even when constitutively active, could not compensate for the absence of Ras proteins. Only constitutive activation of components of the Raf/Mek/Erk pathway was sufficient to sustain normal proliferation and migration of MEFs devoid of Ras proteins. Activation of the phosphatidylinositol 3-kinase (PI3K)/PTEN/Akt and Ral guanine exchange factor (RalGEF)/Ral pathways, either alone or in combination, failed to induce proliferation or migration of Rasless cells, although they cooperated with Raf/Mek/Erk signalling to reproduce the full response mediated by Ras signalling. In contrast to current hypotheses, Ras signalling did not induce proliferation by inducing expression of D-type Cyclins. Rasless MEFs had normal levels of Cyclin D1/Cdk4 and Cyclin E/Cdk2. However, these complexes were inactive. Inactivation of the pocket proteins or knock down of pRb relieved MEFs from their dependence on Ras signalling to proliferate.

The human left and right cerebral hemispheres are anatomically and functionally asymmetric. To test whether human cortical asymmetry has a molecular basis, we studied gene expression levels between the left and right embryonic hemispheres using Serial Analysis of Gene Expression (SAGE), and identified and verified 27 differentially expressed genes, suggesting that human cortical asymmetry is accompanied by early, striking transcriptional asymmetries. LMO4 is consistently more highly expressed in the right perisylvian human cerebral cortex than in the left, and is essential for cortical development in mice, suggesting that human left-right specialization reflects asymmetric cortical development at early stages.One of the most remarkable aspects of the human cerebral cortex is that the two hemispheres are specialized for distinct cognitive and behavioral functions. Whereas the right cerebral cortex regulates movement of the left side of the body and vice versa, approximately 90% of the human population is naturally more skilled with the right hand than with the left (1). This motor asymmetry is strongly correlated with language dominance: language function is predominantly localized to a distributed network in the left perisylvian cortex in 97% of righthanders and about 60% of left-handers (2,3). Functional asymmetries exist in mathematical ability, and spatial and facial recognition as well. These functional asymmetries have been related to anatomical asymmetries of the cortex that are somewhat more subtle (2,4). For example, the posterior end of the Sylvian fissure, is higher in the right hemisphere than in the left (5). The planum temporale, a region in the posterior portion of the superior temporal sulcus in which Wernike's area resides, is larger in the left than in the right in more than 65% of examined adult and 56-79% of fetuses or infant brains, so that the anatomical asymmetries are less striking than the functional ones (6,7). Although genetic factors connecting cerebral Here we directly tested the hypothesis that left-right cortical asymmetry in humans results from differential gene expression at early embryonic stages, long before the onset of organized cerebral cortical function. By applying Serial Analysis of Gene Expression (SAGE), we measured gene expression levels between the left and right hemispheres in early (12-14 weeks) embryonic human brains, during periods of neuronal proliferation and migration, and later (19 weeks), after these processes are largely completed (9). Brain tissues were first dissected from matching perisylvian regions in two hemispheres (Fig. 1, A to C). The cortex was then separated at the midline. On the medial side of the hemisphere, tissues were also dissected from the ventricular zone in the frontal and occipital regions (Fig. 1B). Total RNA was isolated and 14 SAGE libraries were generated (Fig. 1D). To detect genes with differential expression levels, we compared tag frequency for each gene between two SAGE libraries generated from the frontal, perisylvian an...

LMO4 belongs to a family of LIM-only transcriptional regulators, the first two members of which are oncoproteins in acute T cell leukemia. We have explored a role for LMO4, initially described as a human breast tumor autoantigen, in developing mammary epithelium and breast oncogenesis. Lmo4 was expressed predominantly in the lobuloalveoli of the mammary gland during pregnancy. Consistent with a role in proliferation, forced expression of this gene inhibited differentiation of mammary epithelial cells. Overexpression of LMO4 mRNA was observed in 5 of 10 human breast cancer cell lines. Moreover, in situ hybridization analysis of 177 primary invasive breast carcinomas revealed overexpression of LMO4 in 56% of specimens. Immunohistochemistry confirmed overexpression in a high percentage (62%) of tumors. These studies imply a role for LMO4 in maintaining proliferation of mammary epithelium and suggest that deregulation of this gene may contribute to breast tumorigenesis.

The LIM domain is characterized by a double zinc finger structure found in proteins that have critical functions in cell fate determination, growth control, and cytoskeleton organization (reviewed in Refs. 1-4). This motif was originally identified in LIM homeodomain transcription factors and subsequently found in a variety of nuclear and cytoplasmic proteins including LIM-only (LMO), 1 LIM kinase, and focal adhesion proteins. In these proteins, there are usually two or more LIM domains, which may occur in association with functionally divergent domains or by themselves, where they constitute the majority of the protein (1-4). The LIM domain functions primarily as a module for the assembly of protein complexes. There is no evidence to suggest that the LIM domain binds DNA, despite possessing similarity to the GATA-1 zinc finger motifs.The LMO subclass of LIM proteins comprises four members (LMO1-4), each of which is defined by two tandem LIM domains (1, 5). These regulatory molecules appear to have essential functions in cell proliferation and lineage determination. LMO1 and LMO2, both translocated in acute T cell leukemia (T-ALL), are oncogenic within T cells (5). LMO2 has been demonstrated to have a central role in hematopoiesis where it is required for the development of all cell lineages (6). Furthermore, LMO2 has been established to form a multiprotein complex with the hematopoietic transcription factors SCL/TAL-1 and GATA-1 (7-9). These findings indicate a close functional relationship between LMO proteins and DNA-binding factors in blood cells.LMO4, the most recently described member, was isolated by virtue of its interaction with the ubiquitous adaptor protein Ldb1/NL1/CLIM2 (10 -13) and in an expression screen with autologous serum from a breast cancer patient (14 -17). LMO4 is the most divergent member of the LMO subfamily, sharing only 50% homology with the LIM domains of other LMO proteins. The LMO4 gene is widely expressed in embryonic and adult tissues, but high levels are restricted to specific cell types (14,15,17).We have recently established that the LMO4 gene is highly expressed in the proliferating mammary gland during pregnancy and that it is overexpressed in greater than 50% of primary breast cancers (18). Moreover, high levels of LMO4 were found to inhibit mammary differentiation (18). To gain insight into the mechanism by which LMO4 functions in breast epithelium, we searched for partners of LMO4 in these cells. Two interacting proteins were identified, the cofactor CtIP (CtBP-interacting protein) and the breast and ovarian tumor suppressor protein BRCA1, which has previously been shown to associate with . A multiprotein complex involving LMO4, CtIP, and BRCA1 could be demonstrated in vivo. LMO4 was found to be a repressor of BRCA1-mediated transcriptional activity, invoking a potential role for LMO4 as a negative regulator of BRCA1 function in sporadic breast cancers. EXPERIMENTAL PROCEDURESPlasmids-The pGBT9-LMO4 bait plasmid was generated by PCR amplification of mouse LMO4 in pSP72 usi...

The zinc finger protein LMO4 is overexpressed in a high proportion of breast carcinomas. Here, we report that overexpression of a mouse mammary tumor virus (MMTV)-Lmo4 transgene in the mouse mammary gland elicits hyperplasia and mammary intraepithelial neoplasia or adenosquamous carcinoma in two transgenic strains with a tumor latency of 13-18 months. To investigate cellular processes controlled by LMO4 and those that may be deregulated during oncogenesis, we used RNA interference. Down-regulation of LMO4 expression reduced proliferation of human breast cancer cells and increased differentiation of mouse mammary epithelial cells. Furthermore, small-interfering-RNAtransfected breast cancer cells (MDA-MB-231) had a reduced capacity to migrate and invade an extracellular matrix. Conversely, overexpression of LMO4 in noninvasive, immortalized human MCF10A cells promoted cell motility and invasion. Significantly, in a cohort of 159 primary breast cancers, high nuclear levels of LMO4 were an independent predictor of death from breast cancer. Together, these findings suggest that deregulation of LMO4 in breast epithelium contributes directly to breast neoplasia by altering the rate of cellular proliferation and promoting cell invasion.belongs to the LIM-only (LMO) subclass of LIM domain proteins that are defined by two tandem zinc finger domains (1). One of the central functions of the LIM domain is to mediate protein-protein interactions, allowing LMO proteins to act as adaptors for the assembly of multiprotein complexes. LMO proteins have critical roles in normal development. LMO2 is essential for embryonic hematopoiesis and angiogenesis (2, 3), whereas mice lacking both LMO1 and LMO3 die shortly after birth from unknown causes (4). Targeted deletion of Lmo4 (4, 5) results in perinatal lethality accompanied by failure of neural tube closure and homeotic transformations in the rib cage and cervical vertebrae, suggesting that Lmo4 functions as a modulator of Hox protein function.Deregulation of LMO expression can lead to oncogenesis. LMO1 and LMO2 were originally discovered by their association with recurrent translocations in T cell acute lymphocytic leukemia and subsequently were shown to act as T cell oncogenes in transgenic models (1, 6-9). Remarkably, the LMO2 gene was ectopically activated by retroviral integration in severe combined immunodeficient patients who developed T cell leukemia after gene therapy (10). LMO4 was initially identified in an expression screen by using serum from a breast cancer patient (11). Moreover, deregulated expression of LMO4 has been demonstrated in a significant proportion of breast carcinomas (12) and, more recently, in cancers of the oral cavity (13). In breast cancer, LMO4 expression appears to be inversely correlated with estrogen receptor ␣ (ER␣) expression (14). LMO4 can act as a negative regulator of mammary epithelial differentiation in vitro (12), suggesting that LMO4 may have a role in governing cell proliferation. We have shown that LMO4 forms a complex with the corepress...

LMO4 belongs to a family of transcriptional regulators that comprises two zinc-binding LIM domains.LIM-only (LMO) proteins appear to function as docking sites for other factors, leading to the assembly of multiprotein complexes. The transcription factor Deaf-1/NUDR has been identified as one partner protein of LMO4. We have disrupted the Lmo4 and Deaf-1 genes in mice to define their biological function in vivo. All Lmo4 mutants died shortly after birth and showed defects within the presphenoid bone, with 50% of mice also exhibiting exencephaly. Homeotic transformations were observed in Lmo4-null embryos and newborn mice, but with incomplete penetrance. These included skeletal defects in cervical vertebrae and the rib cage. Furthermore, fusions of cranial nerves IX and X and defects in cranial nerve V were apparent in some Lmo4 ؊/؊ and Lmo4 ؉/؊ mice. Remarkably, Deaf-1 mutants displayed phenotypic abnormalities similar to those observed in Lmo4 mutants. These included exencephaly, transformation of cervical segments, and rib cage abnormalities. In contrast to Lmo4 nullizygous mice, nonexencephalic Deaf-1 mutants remained healthy. No defects in the sphenoid bone or cranial nerves were apparent. Thus, Lmo4 and Deaf-1 mutant mice exhibit overlapping as well as distinct phenotypes. Our data indicate an important role for these two transcriptional regulators in pathways affecting neural tube closure and skeletal patterning, most likely reflecting their presence in a functional complex in vivo.The LIM domain is characterized by a double zinc finger structure and is found in proteins that have critical functions in cell fate determination, differentiation, and cytoskeleton organization (reviewed in references 2, 8, and 20). This motif was originally identified in LIM homeodomain transcription factors which have established roles within the central nervous system (CNS). The LIM domain also occurs in a variety of nuclear and cytoplasmic proteins, including LIM-only (LMO), LIM kinase, and focal adhesion proteins. In these proteins, there are usually two or more LIM domains, which may occur by themselves or in association with functionally divergent domains. One of the central functions of the LIM domain is to mediate protein-protein interactions, which may have either positive or negative effects on gene transcription (2, 20).The LMO subclass of LIM proteins comprises four members (LMO1 to LMO4), each of which is defined by two tandem zinc finger domains (30). The LMO1 and LMO2 genes were originally identified by their translocation in acute T-cell leukemia, and their overexpression in transgenic mice leads to T-cell tumors (30). Lmo2 has been established to have a critical function in early hemopoiesis (44) and angiogenesis (43). Little is known about the physiological role of LMO3, which was cloned on the basis of sequence homology. LMO4 was identified by virtue of its interaction with the ubiquitous cofactor protein Ldb1/NLI/CLIM2 (13,21,33) and in an expression screen using autologous serum from a breast cancer patient...

The Ras family of small GTPases constitutes a central node in the transmission of mitogenic stimuli to the cell cycle machinery. The ultimate receptor of these mitogenic signals is the retinoblastoma (Rb) family of pocket proteins, whose inactivation is a required step to license cell proliferation. However, little is known regarding the molecular events that connect Ras signaling with the cell cycle. Here, we provide genetic evidence to illustrate that the p53/ p21 Cdk-interacting protein 1 (Cip1)/Rb axis is an essential component of the Ras signaling pathway. Indeed, knockdown of p53, p21Cip1, or Rb restores proliferative properties in cells arrested by ablation of the three Ras loci, H-, N-and K-Ras. Ras signaling selectively inactivates p53-mediated induction of p21Cip1 expression by inhibiting acetylation of specific lysine residues in the p53 DNA binding domain. Proliferation of cells lacking both Ras proteins and p53 can be prevented by reexpression of the human p53 ortholog, provided that it retains an active DNA binding domain and an intact lysine residue at position 164. These results unveil a previously unidentified role for p53 in preventing cell proliferation under unfavorable mitogenic conditions. Moreover, we provide evidence that cells lacking Ras and p53 proteins owe their proliferative properties to the unexpected retroactivation of the Raf/Mek/Erk cascade by a Ras-independent mechanism.T he RAS genes have been extensively studied due to their key role in mediating mitogenic signaling as well as their high prevalence in human cancers, including those cancers with poor survival rates, such as lung adenocarcinoma, colorectal carcinoma, and pancreatic ductal adenocarcinoma (1, 2). However, the mechanisms by which Ras proteins mediate mitogenic signaling in either normal or tumor cells remain obscure, especially beyond activation of the Raf/Mek/Erk cascade. Recent genetic studies have underscored the relevance of Ras proteins in cellular homeostasis by demonstrating that cells lacking the three Ras loci, H-Ras, N-Ras, and K-Ras (Rasless cells), are completely unable to proliferate (3, 4). Indeed, systemic ablation of these loci in adult mice causes rapid deterioration of multiple tissues, leading to their death in a few days.GTP-loaded Ras proteins promote activation of various downstream signal transduction pathways, mainly the Raf/Mek/Erk kinase cascade, the PI3K/Akt route, and the Ral guanine dissociation stimulator (RalGDS) pathway (1). Activation of other pathways, such as those pathways driven by the Rac family of small G proteins and phospholipase C, has also been illustrated (1). However, genetic interrogation of the pathways essential for cell proliferation has illustrated that only constitutive activation of the Raf, Mek, or Erk kinase can bypass the requirement for Ras proteins to sustain cell division, at least in vitro (3, 4). Indeed, constitutive activation of the PI3K/Akt and RalGDS pathways was incapable of inducing cell proliferation in the absence of Ras proteins. In agreement wit...

LMO2 and LMO4 are members of a small family of nuclear transcriptional regulators that are important for both normal development and disease processes. LMO2 is essential for hemopoiesis and angiogenesis, and inappropriate overexpression of this protein leads to T-cell leukemias. LMO4 is developmentally regulated in the mammary gland and has been implicated in breast oncogenesis. Both proteins comprise two tandemly repeated LIM domains. LMO2 and LMO4 interact with the ubiquitous nuclear adaptor protein ldb1/NLI/CLIM2, which associates with the LIM domains of LMO and LIM homeodomain proteins via its LIM interaction domain (ldb1-LID). We report the solution structures of two LMO:ldb1 complexes (PDB: 1M3V and 1J2O) and show that ldb1-LID binds to the N-terminal LIM domain (LIM1) of LMO2 and LMO4 in an extended conformation, contributing a third strand to a b-hairpin in LIM1 domains. These ®ndings constitute the ®rst molecular de®nition of LIM-mediated protein±protein interactions and suggest a mechanism by which ldb1 can bind a variety of LIM domains that share low sequence homology.