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Figure 6

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Plk1 activates Rock2 in cells. (A) Myc-Rock2 or myc-Rock2-4A was immunoprecipitated from asynchronous cells. Each IP was split in half and one-half has preincubated with Plk1 for 60 min in the presence of cold ATP. Following additional washing, Rock2 kinase activity against myosin regulatory light chain was assayed in the presence of RhoA and [γ-32P]ATP, and analyzed by SDS–PAGE/autoradiography. A strong Rock2 autophosphorylation band is also present. Lanes 1 and 2 in the left panel and lanes 3 and 4 in the right panel are each from the same gel and autoradiograph allowing direct comparison. (B) Overexpression of Rock2 causes accumulation of cells in terminal cytokinesis. Asynchronous U2OS cells were transfected with pEF-BOS (control) or pEF-BOS-expressing wild-type Rock2. Mitotic cells from the control transfections displayed a normal rounded-up morphology, whereas many of the mitotic cells overexpressing Rock2 appeared to be in the terminal midbody stage of cytokinesis. Scale bars=10 μm. (C) Percentage of mitotic cells in cytokinesis 24 h after transfection with myc-Rock2, myc-Rock2-4A, or control vectors. Mean values and s.d. from n=3 experiments. (D) FACS profiles of cells 72 h after transfection with myc-Rock2 or myc-Rock2-4A. (E) Percentage of cells containing greater than 4N DNA content from FACS profiles above. These results are representative of n=3 independent experiments. (F) A model of Plk1 control of regulatory myosin light chain (RMLC) phosphorylation during cytokinesis. Arrows indicate activation events and bars indicate inhibition events. Kinases are indicated as squares, phosphatases as circles, GEFs as diamonds, and small G-proteins as rounded squares.

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