Beta1-class integrins regulate the development of laminae and folia in the cerebral and cerebellar cortex.

Mice that lack all beta1-class integrins in neurons and glia die prematurely after birth with severe brain malformations. Cortical hemispheres and cerebellar folia fuse, and cortical laminae are perturbed. These defects result from disorganization of the cortical marginal zone, where beta1-class integrins regulate glial endfeet anchorage, meningeal basement membrane remodeling, and formation of the Cajal-Retzius cell layer. Surprisingly, beta1-class integrins are not essential for neuron-glia interactions and neuronal migration during corticogenesis. The phenotype of the beta1-deficient mice resembles pathological changes observed in human cortical dysplasias, suggesting that defective integrin-mediated signal transduction contributes to the development of some of these diseases.

p45SKP2 promotes p27Kip1 degradation and induces S phase in quiescent cells.

The F-box protein p45SKP2 is the substrate-targeting subunit of the ubiquitin-protein ligase SCFSKP2 and is frequently overexpressed in transformed cells. Here we report that expression of p45SKP2 in untransformed fibroblasts activates DNA synthesis in cells that would otherwise growth-arrest. Expression of p45SKP2 in quiescent fibroblasts promotes p27Kip1 degradation, allows the generation of cyclin-A-dependent kinase activity and induces S phase. Coexpression of a degradation-resistant p27Kip1 mutant suppresses p45SKP2-induced cyclin-A-kinase activation and S-phase entry. We propose that p45SKP2 is important in the progression from quiescence to S phase and that the ability of p45SKP2 to promote p27Kip1 degradation is a key aspect of its S-phase-inducing function. In transformed cells, p45SKP2 may contribute to deregulated initiation of DNA replication by interfering with p27Kip1 function.

Multimerization of polyomavirus middle-T antigen.

The oncogenic protein of polyomavirus, middle-T antigen, associated with cell membranes and interacts with a variety of cellular proteins involved in mitogenic signalling. Middle-T antigen may therefore mimic the function of cellular tyrosine kinase growth factor receptors, like the platelet-derived growth factor or epidermal growth factor receptor. Growth factor receptor signalling is initiated upon the binding of a ligand to the extracellular domain of the receptor. This results in activation of the intracellular tyrosine kinase domain of the receptor, followed by receptor phosphorylation, presumably as a consequence of dimerization of two receptor molecules. Similar to middle-T antigen, phosphorylation of growth factor receptors leads to recruitment of cellular signalling molecules downstream in the signalling cascade. In this study, we investigated whether middle-T antigen, similar to tyrosine kinase growth factor receptors, is able to form dimeric signalling complexes. We found that association with cellular membranes was a prerequisite for multimerization, most likely dimer formation. A chimeric middle-T antigen carrying the membrane-targeting sequence of the vesicular stomatitis virus G protein instead of the authentic polyomavirus sequence still dimerized. However, mutants of middle-T antigen unable to associate with 14-3-3 proteins, like d18 and S257A, did not form dimers but were still oncogenic. This indicates that both membrane association and binding of 14-3-3 are necessary for dimer formation of middle-T antigen but that only the former is essential for cell transformation.

A role for the small GTPase Rac in polyomavirus middle-T antigen-mediated activation of the serum response element and in cell transformation.

The oncogenic proteins encoded by papovaviruses, the tumor antigens, have been extensively used as model systems to study mitogenic signaling and cell transformation. These proteins stimulate cell growth in cultured cells and induce tumors in virus infected or transgenic animals. One of these proteins, polyomavirus middle-T, acts like a constitutively activated tyrosine growth factor receptor. Middle-T recruits several cellular enzymes into a multifunctional complex located at cellular membranes. This results in the activation of cellular enzymes involved in the regulation of cell signaling, like tyrosine kinases of the Src family, a phosphatidylinositol 3-kinase and a GDP/GTP exchange factor for Ras. These activities are all required for stimulation of cell growth by middle-T and activate members of the MAP kinase family. Here we investigate the role of T antigen-activated pathways in the stimulation of transcription of immediate early genes. These genes are essential for progression of resting cells into S phase. Our data show that Rho family GTPases play an essential role in cell transformation by middle-T. Furthermore, we demonstrate that the c-fos promoter is activated by two Ras-initiated signaling cascades. One is Raf-dependent and requires binding of SHC and PI 3-kinase to the middle-T complex. This pathway signals via ternary complex factor (TCF) to the serum response element (SRE) of the c-fos promoter. Signaling to TCF by Raf also depends on functional Rac, but not CDC42, as demonstrated in luciferase reporter assays with an ETS domain-containing promoter. The second pathway is Raf-independent, does not require SHC but functional PI 3-kinase, and transduces signals via Rac to serum response factor (SRF). Microinjection of dominant negative Rac1 blocks nuclear translocation of ERK1 in middle-T-expressing cells. This lends support to the idea that the two signaling cascades initiated by Ras show crosstalk at the level of MAP kinase-mediated signaling to nuclear transcription factors.

Polyomavirus middle-T antigen associates with the kinase domain of Src-related tyrosine kinases.

Middle-T antigen of mouse polyomavirus, an oncogenic DNA virus, associates with and activates the cellular tyrosine kinases c-Src, c-Yes, and Fyn. This interaction is essential for polyomavirus-mediated transformation of cells in culture and tumor formation in animals. To determine the domain of c-Src directing association with middle-T, mutant c-Src proteins lacking the amino-terminal unique domain and the myristylation signal, the SH2 domain, the SH3 domain, or all three of these domains were coexpressed with middle-T in NIH 3T3 cells. All mutants were found to associate with middle-T, demonstrating that the kinase domain of c-Src, including the carboxy-terminal regulatory tail, is sufficient for association with middle-T. Moreover, we found that Hck, another member of the Src kinase family, does not bind middle-T, while chimeric kinases consisting of the amino-terminal domains of c-Src fused to the kinase domain of Hck or the amino-terminal domains of Hck fused to the kinase domain of c-Src associated with middle-T. Hck mutated at its carboxy-terminal regulatory residue, tyrosine 501, was also found to associate with middle-T. These results suggest that in Hck, the postulated intramolecular interaction between the carboxy-terminal regulatory tyrosine and the SH2 domain prevents association with middle-T. This intramolecular interaction apparently also limits the ability of c-Src to associate with middle-T, since removal of the SH2 or SH3 domain increases the efficiency with which middle-T binds c-Src.

Domains in middle-T antigen that cooperate in polyomavirus-mediated oncogenic transformation.

Middle-T antigen is the oncogenic protein of Polyomavirus and associates with several cellular enzymes involved in signal transduction, e.g., Src tyrosine kinases, phosphatidylinositol 3-kinase (PI 3-kinase), protein phosphatase 2A (PP2A), and Shc, an SH2 domain-containing adapter protein. We have shown earlier that middle-T is a target of a cell cycle-regulated serine/threonine-specific kinase, presumably p34cdc2. Phosphorylation of middle-T by p34cdc2 results in increased apparent M, weight of the protein on SDS-polyacrylamide gels. Two threonine residues in positions 160 and 291, respectively, were identified in the middle-T sequence as putative targets of a cyclin-dependent kinase. Replacement of threonine 160 by alanine resulted in a transformation-defective mutant protein that was still capable of forming all the complexes with cellular proteins, suggesting that additional characteristics of middle-T are required for cell transformation. In the present study we report that the defect of the T160A middle-T mutant is compensated by mutations introduced into a domain encompassing amino acids 253 to 302. In particular, mutating serine 283, a canonical phosphorylation site for a cyclin-dependent kinase, to an alanine residue rendered the T160A middle-T mutant wild type. Based on these results we suggest that cell cycle-specific phosphorylation of specific serine and threonine residues by cyclin-dependent kinases regulates middle-T function.

Catalytic activity and transformation potential of v-Src require arginine 385 in the substrate binding pocket.

Tyrosine kinase are important mediators of signal transduction in eukaryotic cells. In order to better understand the mechanism of catalysis we studied a set of mutants of the prototype tyrosine kinase, the c-Src protein, a homologue of the Rous Sarcoma virus oncogene. Based on an X-ray structure of cAMP-dependent protein kinase (cAPK) we mutated an arginine residue conserved in subdomain VI of all known kinases to a non-charged residue. This residue coordinates phosphate of the autophosphorylation site located in subdomain VII of cAPK and this interaction has been proposed to be crucial for substrate binding. The mutant R385A of c-Src had low kinase activity towards exogenous substrates yet was able to autophosphorylate at tyrosine 416. When introduced into an activated v-src gene the R385A mutation totally blocked cell transformation. Our data suggest that the function of the conserved arginine 385 is to coordinate the phosphate of the autophosphorylation site and to provide in this way a stable template for substrate binding.

Mitosis-specific phosphorylation of polyomavirus middle-sized tumor antigen and its role during cell transformation.

Transformation of cells in culture by polyomavirus is mediated by one of its early gene products, middle-sized tumor antigen (MTAg). This protein forms multiple complexes with cellular enzymes such as tyrosine kinases (pp60c-src), a phosphatidylinositol 3-kinase, and phosphatase 2A. Association with MTAg leads to the activation of pp60c-src through interference with phosphorylation at Tyr-527, a site negatively regulating src kinase activity. MTAg abrogates mitosis-specific activation of pp60c-src, resulting in constitutive high kinase activity of the enzyme throughout all phases of the cell cycle. Here we report that MTAg is transiently modified during mitosis, resulting in an increase in its apparent molecular size on SDS/acrylamide gels. Similarly, MTAg isolated from interphase cells and phosphorylated by the cell cycle-regulated serine/threonine kinase p34cdc2 in vitro has increased molecular mass. The large molecular mass form of the protein can be converted to the authentic 56-kDa form upon dephosphorylation by potato acid phosphatase. Two putative phosphorylation sites for a cdc2-like kinase were identified as Thr-160 and -291, respectively. Conversion of Thr-160 to Ala resulted in a transformation-defective mutant protein that was still capable of associating with pp60c-src, phosphatidylinositol 3-kinase, and phosphatase 2A, while the corresponding mutant in position 291 was wild type with respect to all parameters measured so far. These data suggest that phosphorylation by p34cdc2 or a related cell cycle-regulated kinase modulates the interaction of MTAg with cellular targets that are crucial for cell transformation.