The human tumour suppressor LATS1 is activated by human MOB1 at the membrane
Section snippets
Materials and methods
Cell culture, chemicals, drug treatments, transfections, and antibodies. COS-7 cells were cultured and transfected as described [11]. In some experiments, cells were treated for 60 min with 1 μM okadaic acid (OA; Alexis Corp.). In other experiments, cells were serum-starved for 2 h prior to transfection. The transfection mixture was removed after 4 h and cells were serum-starved overnight before stimulation with 12-O-tetradecanoylphorbol 13-acetate (TPA; 100 ng/ml; Amersham Biosciences). Anti-HA
Human LATS1 kinase activity can be measured using the substrate peptide of human NDR1/2
Since LATS kinase activity has previously only been monitored by autophosphorylation [2], [39], we established a kinase assay to monitor human LATS1 transphosphorylation activity. By applying the same conditions as reported for human NDR1/2 kinases [11], we could readily measure human LATS1 kinase activity towards the standard NDR1/2 substrate peptide (Fig. 1). As predicted, HA-LATS1 immunoprecipitates displayed elevated kinase activity once HA-LATS1 wild-type (wt) expressing cells had been
Discussion
In summary, our interaction data are in full agreement with recent observations [14], by showing that LATS1 associates with hMOB1A and hMOB1B, but not hMOB2. Further, we show that human LATS1 and NDR1/2 share a very similar hMOB1-binding motif through which kinase activity is regulated. The hMOB1-binding motif also exists in human LATS2 and even in Lats from flies (see Fig. 3A), predicting that mutations at key residues defined in our study will also affect their interaction with MOBs. By
Acknowledgments
We thank M. Pietrzak for sequencing and F. Fischer for synthesis of peptides. Special thanks go to H.H. Sillje for providing LATS1 (wt) and (D846A) cDNAs. We are also grateful to J. Lisztwan and P. King for careful reading of the manuscript. This work was supported by the Swiss Cancer League Grant KLS-01342-02-2003. D. Schmitz was supported by the Boehringer Ingelheim Fonds. The Friedrich Miescher Institute is part of the Novartis Research Foundation.
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