Abstract The non-receptor spleen tyrosine kinase Syk has mainly been studied in hematopoietic cells in which it plays a key role in the immune-response signalling. We previously demonstrated that Syk is also present in mammary epithelial cells and that its expression is lost during malignant progression of breast cancer cell lines. Clinical studies confirmed loss of Syk expression, not only in breast tumors but also in other carcinomas and melanoma. Xenotransplantation animal experiments evidenced that Syk acts as a tumor and metastasis suppressor in breast cancer and melanoma. The mechanisms by which Syk exerts its anti-oncogenic activity remain, however, unrevealed. We previously demonstrated that Syk localizes to the centrosomes and negatively affects cell division. The centrosomal Syk substrates and possible effectors of its anti-oncogenic activity remain, nevertheless, unknown. In this study, we demonstrate that Syk may negatively affect cell cycle progression through the phosphorylation of the Cdk1 kinase, a key protein involved in the control of mitosis. Using immunofluorescence, we observed that endogenous Syk and Cdk1 colocalize at the centrosomes and that transient DsRed-Syk transfection induces pY15-Cdk1-positive epitopes at the centrosomes. In vitro kinase assays demonstrate that Syk can phosphorylate Cdk1 in the presence of different cyclins. Mass spectrometry allowed identification of four different Cdk1 tyrosine residues phosphorylated by Syk, amongst which the Y15 is known to negatively affect mitotic entry when phosphorylated by the Wee1 kinase. Transient overexpression of DsRed-Syk or GFP-Cdk1 carrying phospho-mimicking mutations resulted in a cell cycle block as observed by FACS analysis and led to a senescence-like growth arrest. Finally, we demonstrate that DNA damage-inducing agents induce Syk activation and Cdk1 tyrosine phosphorylation in a Syk-dependent manner, as evidenced by the use of a Syk-targeting shRNA. In conclusion, our results reveal that Syk overexpression or Syk activation by genotoxic agents induce a cell cycle arrest through the phosphorylation of Cdk1. These novel aspects of Syk function will contribute to a better understanding of its onco-suppressive activity and, more generally, of the DNA damage-induced signalling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2959. doi:10.1158/1538-7445.AM2011-2959