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. 2010 Mar 29;6(2):163-71.
doi: 10.7150/ijbs.6.163.

The ulcerative colitis marker protein WAFL interacts with accessory proteins in endocytosis

You Fu Pan  1 , Ing-Marie Viklund, Heng Hang Tsai, Sven Pettersson, Ichiro N Maruyama
Affiliations

The ulcerative colitis marker protein WAFL interacts with accessory proteins in endocytosis

You Fu Pan et al. Int J Biol Sci. .

Abstract

Ulcerative colitis (UC) is one of the major forms of inflammatory bowel disease with unknown cause. A molecular marker, WAFL, has recently been found to be up-regulated in the inflamed colonic mucosa of UC patients. Towards understanding biological function of WAFL, we analyzed proteins interacting with WAFL in HEK-293 cells by immunoprecipitation and mass spectrometry. Among four proteins found to specifically interact with WAFL, both KIAA0196 and KIAA1033 bind to alpha-appendage of the adaptor protein complex 2 (AP2), which acts as an interaction hub for accessory proteins in endocytosis mediated by clathrin-coated vesicle (CCV). The specific interaction between WAFL and KIAA0196 was also confirmed in human colorectal carcinoma HCT-116 cells by co-immunoprecipitation with specific antibodies. Meta-analyses of the databases of expressed genes suggest that the three genes are co-expressed in many tissues and cell types, and that their molecular function may be classified in the category of 'membrane traffic protein'. Therefore, these results suggest that WAFL may play an important role in endocytosis and subsequent membrane trafficking by interacting with AP2 through KIAA0196 and KIAA1033.

Keywords: Inflammatory bowel disease; KIAA0196/strumpellin; KIAA1033; Proteomics; WAFL/FKBP15/FKBP133/KIAA0674; Wiskott-Aldrich syndrome protein.

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Conflict of interest statement

Conflict of Interest: The authors have declared that no conflict of interest exists.

Figures

Fig 1
Fig 1
(A) Western blot analysis of the WAFL protein fused with a FLAG tag. HEK-293 cells were transfected with an expression plasmid encoding the WAFL-FLAG fusion. The cells were lysed, and then immunoprecipitated with anti-FLAG antibody. Proteins before and after the immuntoprecipitation were resolved by SDS-PAGE. After the electrophoresis, proteins were transferred onto a membrane filter and detected with anti-FLAG antibody. Lane #1: immunoprecipitate from cells transfected; #2: immunoprecipitate from cells untransfected; #3: total proteins from cells transfected; #4: total proteins from cells untransfected. Positions of molecular standards are also shown on the right margin. (B) Proteins co-immunoprecipitated with the WAFL-FLAG fusion protein by using immobilized anti-FLAG antibody. After separating eluates from the resin by SDS-PAGE, proteins were silver-stained. Lane #1: immunopriciptate from cells transfected; #2: immunopriciptate from cells untransfected; #3: immunoprecipitate from lysis buffer (negative control); #4: eluate from anti-FLAG antibody resin used for the immunoprecipitation (negative control). Protein bands on the lane #1 that were not observed in lane #2 are indicated by arrows with numbers on the left margin. Positions of molecular standards are also shown on the right margin. (C) Co-immunoprecipitation (IP) of the KIAA0196 protein with antibody specific for WAFL. Human HCT-116 cell lysate was reacted with antibody specific for WAFL, and the resulting complex was precipitated with protein A-Sepharose. The protein complex was eluted from the Sepharose beads, was resolved by SDS-PAGE, and was analyzed by Western blotting (WB) with antibody specific for KIAA0196. (D) Co-immunoprecipitation of WAFL with antibody specific for the KIAA0196 protein.
Fig 2
Fig 2
Representative CID fragmentation profiles are given for interacting proteins of WAFL/KIAA0674 identified by LC-MS/MS. (A) CID fragmentation profile of CLALQAQITALTK derived from WAFL/KIAA0674 (T00363). The monoisotopic mass of the neutral peptide is 1429.80 Da. Ions score is 59. b(2)-b(12) and y(2)-y(11) are matched with the peptide shown in the insert. (B) CID fragmentation profile of EEMVLDNIPK derived from KIAA0196 (Q53EL1_HUMAN). The monoisotopic mass of the neutral peptide is 1186.59 Da. Ions score is 39. b(2)-b(9) and y(2)-y(9), except b(7), are matched with the peptide shown in the insert. (C) CID fragmentation profile of FLEEYTSQLR derived from KIAA1033 (Q2M389). The monoisotopic mass of the neutral peptide is 1284.63 Da. Ions score is 44. b(2)-b(9) and y(2)-y(8) are continuously detected and matched with the peptide shown in the insert.
Fig 3
Fig 3
Meta-analysis of WAFL and its interacting proteins. (A) Correlation network of WAFL and its interacting partners. Solid lines indicate the strongest correlation among all the combinations with partner genes, based on co-expression frequencies of corresponding genes in the database, and dashed lines imply statistically significant, but less frequent than that of the strongest, correlations. (B-D) Gene ontology analysis of WAFL and its interacting partners. Top 1,000 genes co-expressed were selected from the PANTHER database, and statistically significantly (p < 0.01) enriched ontology categories were identified as molecular functions. The following three gene sets were analyzed: (B) WAFL, (C) KIAA0196, and (D) KIAA1033. Note that the 'membrane traffic protein' category is commonly enriched.
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