Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2011 Jan 15;22(2):266–277. doi: 10.1091/mbc.E10-07-0605

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© 2011 Ishitani et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

“ASCB®,“ “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society of Cell Biology.

PMC Copyright notice

FIGURE 4: — NLK forms homo-oligomers dependent on Cys-425. (A) NLK forms homo-oligomers in vivo. HEK293 cells were transfected with HA-NLK (WT), HA-NLK(K155M) (KM), HA-NLK(C425Y) (CY), Flag-NLK (WT), Flag-NLK(K155M), Flag-NLK(T286V) (TV), and Flag-NLK(C425Y) (CY) as indicated. Cell extracts were subjected to immunoprecipitation with anti-Flag antibody. Immunoprecipitated complexes were immunoblotted with anti-HA (top) and anti-Flag (bottom) antibodies. Expression of HA-NLK was confirmed by immunoblotting with anti-HA antibody (middle). Asterisk indicates IgG. (B) NLK forms homo-oligomers in vitro. HEK293 cells were transfected with Flag-NLK (WT), NLK(C425Y) (CY), and NLK(1–415) (N-KD). The cell extracts were mixed with cell extracts prepared from cells transfected with HA-NLK (WT), NLK(C425Y) (CY), and NLK(1–415) (N-KD) and then immunoprecipitated with anti-Flag antibody. Immunoprecipitated complexes were immunoblotted with anti-HA (top left) and anti-Flag (bottom left) antibodies. Expression of HA-NLK proteins in cell extracts was confirmed by immunoblotting with anti-HA antibody (right). (C) Schematic diagram of NLK deletion mutants. (D) Both the kinase and C-terminal domains of NLK are required for oligomerization. HEK293 cells were transfected with HA-NLK(K155M) (KM) and Flag-NLK variants as indicated. Cell extracts were subjected to immunoprecipitation with anti-Flag antibody. Immunoprecipitated complexes were immunoblotted with anti-HA and anti-Flag antibodies. Expression of HA-NLK(K155M) was confirmed by immunoblotting with anti-HA antibody (middle). (E) The oligomerization-defective NLK mutant lacks autophosphorylation activity. Flag-NLK (Full) and NLK(1–415) (N-KD) were expressed in HEK293 cells, and the proteins were immunopurified. Aliquots of purified NLK were subjected to non-RI in vitro kinase assay with or without ATP and then immunoblotted with anti-Flag and anti-pNLK antibodies. (F and G) The oligomerization-defective NLK mutant lacks Tcf4 phosphorylation activity in vitro (F) and in vivo (G). (F) Flag-NLK (Full) and NLK(1–415) (N-KD) were expressed in HEK293 cells, and the proteins were immunopurified. Flag-Tcf4 was also expressed in HEK293 cells, and the proteins were purified. Aliquots of purified Tcf4 and NLK proteins were subjected to non-RI in vitro kinase assay and then immunoblotted with anti-Flag antibody (top). Purified NLK proteins were also confirmed by immunoblotting with anti-Flag antibody (bottom). (G) HEK293 cells were transfected with Flag-NLK (Full), NLK(1–415) (N-KD), and Flag-Tcf4, and then lysates were immunoblotted with anti-Flag antibody. (H) Substitution of Cys-425 with tyrosine, but not serine, reduces the Tcf4 substrate–phosphorylating activity of NLK. HEK293 cells were transfected with Flag-NLK(WT), NLK(C425Y) (CY), NLK(C425S) (CS), and Flag-Tcf4, and then lysates were immunoblotted with anti-Flag antibody.