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Fig. 4. NIP2 was phosphorylated by Nek2 in vitro and in vivo. (A) In vitro kinase assay. The wild-type (Nek2RHA1) and kinase-defective (Nek2KHA5) Nek2 proteins were prepared from transiently transfected 293T cells. As substrates, GST-NIP2^1-523 was purified from bacterial lysates and Myc-NIP2^1-523 was prepared by immunoprecipitation from transfected 293T cells. Casein was a control substrate for Nek2. Specific bands on the autoradiogram are marked. The bands marked with an asterisk are non-specific. (B) In vitro kinase assay carried out with wild-type (WT) or kinase-defective (KD) Nek2. The substrates were prepared from bacterially expressed GST fusion proteins linked to NIP2^1-193, NIP2^194-560 or NIP2^561-903. The separated gel was stained with Coomassie Blue and autoradiographed. The arrowheads indicate the substrate proteins and the asterisk indicates autophosphorylation of Nek2. (C) In vivo labeling of NIP2. HeLa cells transfected with pNek2RHA1 or pNek2KHA5 were incubated in the presence of ³²P-orthophosphate (250 µCi/ml) for 3 hours. Endogenous NIP2 was immunoprecipitated (IP) and subjected to immunoblot analysis as well as autoradiogram. Exogenous Nek2 was also immunoprecipitated with the HA antibody, followed by immunoblot analysis and autoradiogram. A faint band of HA immunoprecipitate from untransfected cells corresponds to the IgG heavy chain.

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