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. 2004 Apr 1;18(7):816–829. doi: 10.1101/gad.1170604

Figure 2.

Figure 2.

HIPK2 functions between TAK1 and NLK in the signaling pathway. (A) NLK is coimmunoprecipitated with HIPK2. 293 cells were transfected with expression vectors encoding Flag-tagged wild-type (WT) or kinase-negative mutant (K221R) HIPK2 together with HA-NLK. Cell lysates were immunoprecipitated (IP) with anti-Flag antibody, and the immunocomplexes were analyzed by Western blotting with anti-HA antibody (top) or anti-Flag antibody (middle). (Bottom) The amount of HA-NLK in the whole cell extracts is also shown. (B) HIPK2 phosphorylates NLK. 293 cells were transfected with expression vectors for Flag-HIPK2 together with HA-tagged kinase-negative NLK (HA-NLK-KN). (Top) Cell lysates were immunoprecipitated with anti-Flag antibody, and the immunocomplexes were subjected to the in vitro kinase assay. The amount of HA-NLK-KN in the anti-Flag immunoprecipitates (middle) and the whole-cell extracts (bottom) are shown. (C) HIPK2 enhances NLK kinase activity. 293 cells were transfected with expression vectors for wild-type or kinase-negative HA-NLK together with various amounts of Flag-HIPK2. (Top) Cell lysates were immunoprecipitated with anti-HA antibody and subjected to the in vitro kinase assay with bacterially expressed GST–LEF-1 as an exogenous substrate. (Bottom) The amount of immunoprecipitated HA-NLK is shown. (D) HIPK2 does not phosphorylate the site in the T-loop of NLK. 293 cells were transfected with expression vectors for kinase-negative (K155M) HA-NLK containing the normal or mutated (T286V) T-loop together with Flag-HIPK2. (Top) Cell lysates were immunoprecipitated with anti-HA antibody and subjected to the in vitro kinase assay. (Bottom) The amounts of immunoprecipitated HA-NLK and Flag-HIPK2 are shown. (E) TAK1 phosphorylates HIPK2. 293 cells were transfected with the expression vectors for TAB1 and HA-tagged wild-type (WT) or kinase-negative mutant (KN) TAK1 and immunoprecipitated with anti-HA antibody. The immunoprecipitates were subjected to an in vitro kinase assay using bacterially expressed GST–HIPK2 as a substrate.