In normal and transformed cells, the F‐box protein p45SKP2 is required for S phase and forms stable complexes with p19SKP1 and cyclin A–cyclin‐dependent kinase (CDK)2. Here we identify human CUL‐1, a member of the cullin family, and the ubiquitin‐conjugating enzyme CDC34 as additional partners of p45SKP2 in vivo . CUL‐1 also associates with cyclin A and p19SKP1 in vivo and, with p45SKP2, they assemble into a large multiprotein complex. In Saccharomyces cerevisiae , a complex of similar molecular composition (an F‐box protein, a member of the cullin family and a homolog of p19SKP1) forms a functional E3 ubiquitin protein ligase complex, designated SCFCDC4, that facilitates ubiquitination of a CDK inhibitor by CDC34. The data presented here imply that the p45SKP2–CUL‐1–p19SKP1 complex may be a human representative of an SCF‐type E3 ubiquitin protein ligase. We propose that all eukaryotic cells may use a common ubiquitin conjugation apparatus to promote S phase. Finally, we show that multiprotein complex formation involving p45SKP2–CUL‐1 and p19SKP1 is governed, in part, by periodic, S phase‐specific accumulation of the p45SKP2 subunit and by the p45SKP2‐bound cyclin A–CDK2. The dependency of p45SKP2–p19SKP1 complex formation on cyclin A–CDK2 may ensure tight coordination of the activities of the cell cycle clock with those of a potential ubiquitin conjugation pathway.

Sprouty (Spry) proteins function as inhibitors of receptor tyrosine kinase signaling mainly by interfering with the Ras/Raf/mitogen-activated protein kinase cascade, a pathway known to be frequently deregulated in human non–small cell lung cancer (NSCLC). In this study, we show a consistently lowered Spry2 expression in NSCLC when compared with the corresponding normal lung epithelium. Based on these findings, we investigated the influence of Spry2 expression on the malignant phenotype of NSCLC cells. Ectopic expression of Spry2 antagonized mitogen-activated protein kinase activity and inhibited cell migration in cell lines homozygous for K-Ras wild type, whereas in NSCLC cells expressing mutated K-Ras, Spry2 failed to diminish extracellular signal-regulated kinase (ERK) phosphorylation. Nonetheless, Spry2 significantly reduced cell proliferation in all investigated cell lines and blocked tumor formation in mice. Accordingly, a Spry2 mutant unable to inhibit ERK phosphorylation reduced cell proliferation significantly but less pronounced compared with the wild-type protein. Therefore, we conclude that Spry2 interferes with ERK phosphorylation and another yet unidentified pathway. Our results suggest that Spry2 plays a role as tumor suppressor in NSCLC by antagonizing receptor tyrosine kinase–induced signaling at different levels, indicating feasibility for the usage of Spry in targeted gene therapy of NSCLC. (Mol Cancer Res 2007;5(5):509–20)