Early insights into the function of KIAA1199, a markedly overexpressed protein in human colorectal tumors

Abstract

We previously reported that the expression of KIAA1199 in human colorectal tumors (benign and malignant) is markedly higher than that in the normal colonic mucosa. In this study, we investigated the functions of the protein encoded by this gene, which are thus far unknown. Immunostaining studies were used to reveal its subcellular localization, and proteomic and gene expression experiments were conducted to identify proteins that might interact with KIAA1199 and molecular pathways in which it might play roles. Using colon cancer cell lines, we showed that both endogenous and ectopically expressed KIAA1199 is secreted into the extracellular environment. In the cells, it was found mainly in the perinuclear space (probably the ER) and cell membrane. Both cellular compartments were also over-represented in lists of proteins identified by mass spectrometry as putative KIAA1199 interactors and/or proteins encoded by genes whose transcription was significantly changed by KIAA1199 expression. These proteomic and transcriptomic datasets concordantly link KIAA1199 to several genes/proteins and molecular pathways, including ER processes like protein binding, transport, and folding; and Ca(2+), G-protein, ephrin, and Wnt signaling. Immunoprecipitation experiments confirmed KIAA1199's interaction with the cell-membrane receptor ephrin A2 and with the ER receptor ITPR3, a key player in Ca(2+) signaling. By modulating Ca(2+) signaling, KIAA1199 could affect different branches of the Wnt network. Our findings suggest it may negatively regulate the Wnt/CTNNB1 signaling, and its expression is associated with decreased cell proliferation and invasiveness.

Figure 1. Expression and localization of KIAA1199.

A. Western blots showing KIAA1199 protein levels in distinct subcellular compartments of LS174T cells and in conditioned medium. CDH1 (E-cadherin), CANX (calnexin), TUBB, and TFIIH were used as cell-membrane, ER, cytoplasmic, and nuclear markers, respectively. B. Immunocytochemical localization of KIAA1199 in LS174T (upper panel) and SW480 cells (lower panel). Gray arrowheads: cell membrane expression; green arrowheads: perinuclear expression. Inset: negative control (immunostaining without primary antibody). C. Constitutive and inducible expression of KIAA1199 in transfected SW480 cells. Top: SW480 clones expressing KIAA1199 constitutively. Dest-V5, cells transfected with empty vector pcDNA3.2V5DEST; KIAA1199-V5, cells expressing KIAA1199-V5 tagged at C-terminus; KIAA1199-Cl.18, cells expressing untagged KIAA1199. Bottom: SW480 clones expressing KIAA1199 upon doxycycline induction. TR, tetracycline-repressor-expressing control cells; KIAA1199-Cl.13, cells expressing KIAA1199 only in the presence of doxycycline; KIAA1199-GFP, cells expressing C-terminal GFP-tagged KIAA1199 only in the presence of doxycycline. The total cell extract of LS174T cells (endogenous KIAA1199 expressors) was used as a positive control; TUBB was used as loading control. D. Subcellular localization of KIAA1199 in SW480 KIAA1199-V5 cells. CDH1, CANX, TUBB, and TFIIH were used as cell-membrane, ER, cytoplasmic, and nuclear markers, respectively. E. Immunocytochemical staining of SW480 KIAA1199-V5 cells shows KIAA1199 in the cell membrane (gray arrowheads) and perinuclear space (green arrowheads). Upper right inset: Fine focusing clearly reveals staining of the nuclear membrane (red arrowheads). Lower right inset: Negative control (SW480 Dest-V5 immunostained with V5-tag-specific antibody).

Figure 2. KIAA1199 interactome and gene expression changes associated with KIAA1199 expression in SW480 cells.

A. The table shows cell lines and antibodies used in the three immunoprecipitation experiments performed to identify proteins that interact with KIAA1199. B. Representative Western blots showing the results of the immunoprecipitation experiments summarized in the table. Input: exp.1 and 3, KIAA1199-V5 extract; exp.2, KIAA1199-Cl.18 extract. C. Silver-stained gel showing unique bands present only in the KIAA1199 immunoprecipitate (black asterisk) following IP with antibodies against the V5 tag. (The immunoprecipitated KIAA1199 is indicated with a red asterisk.) D. Upper four strips: Western blot showing co-immunoprecipitation of ITPR3 with anti-KIAA1199 antibody and reciprocal co-immunoprecipitation of KIAA1199 with anti-ITPR3 antibody. Lower four strips: Western blot showing co-immunoprecipitation of EPHA2 with anti-KIAA1199 antibody (arrowheads) and reciprocal co-immunoprecipitation of KIAA1199 with anti-EPHA2 antibody. Input: KIAA1199-Cl.18 extract. The weak interactions of these two proteins with KIAA1199 might be related to the fact that IP was performed using extracts from cells in which these two proteins were endogenously expressed (instead of overexpressed). E. Top: Western blot showing KIAA1199 protein levels in SW480 Clone 13 total cell extracts and in conditioned medium before and after doxycycline induction. TUBB: loading control. Bottom: Principal component analysis (PCA) of log₂ KIAA1199 mRNA expression intensity values. The plot of PCA scores for the six samples 48 h after doxycycline induction shows clear separation of replicates with (n = 3) vs. without (n = 3) KIAA1199 expression. The first three principal components explain 72.9% of the total variance. F. Heatmap showing expression of the 490 differentially expressed genes (y axis) before and after doxycycline-induced KIAA1199 expression in three replicate experiments. (Green: upregulated; red: downregulated in KIAA1199-expressing cells; p value <0.025, and fold change >1.2).

Figure 3. KIAA1199 exerts negative feedback on canonical Wnt signaling.

A. Ectopic KIAA1199 expression in HEK293 cells inhibits the transcriptional activity elicited by wild-type CTNNB1 or by a constitutively active form of the protein, CTNNB1-T41A. Cells were transiently transfected with either a TOPflash or a FOPflash reporter vector, together with an empty vector or an expression vector for KIAA1199 or TCF4-DN. TOP/FOP ratios are the mean ± SEM of triplicate experiments. B. Total and active CTNNB1 levels in SW480 cells expressing KIAA1199 (V5) and in empty vector-transfected (Dest) controls. The table shows fold changes (vs. controls) in the expression of both CTNNB1 forms induced by KIAA1199 expression based on quantification of band intensity relative to that of CDH1 or TUBB in the same lane. TUBB was used as loading control for cytoplasmic and nuclear fractions since it was detectable in these fractions of the SW480-V5 and Dest cell extracts under the same extraction conditions. Furthermore, TUBB has been reported to shuttle to the nucleus ( and references herein).

Figure 4. Ectopic expression of KIAA1199 decreases cytoplasmic and nuclear levels of CTNNB1.

A. Immunocytochemical staining for total and active CTNNB1 in empty vector-transfected SW480 cells (Dest-V5) and SW480 cells expressing KIAA1199-V5 constitutively. The latter cells, presented decreased CTNNB1 levels in the nucleus and cytoplasm. B. Similar results were observed in SW480 KIAA1199-GFP cells (immunofluorescence experiments). Although only a fraction of these cells exhibited substantial KIAA1199-GFP expression upon doxycycline induction, the vast majority displayed CTNNB1 depletion (total and active forms) in both the nucleus and cytoplasm, which is probably a paracrine effect of secreted KIAA1199.

Figure 5. Effect of ectopic constitutive KIAA1199 expression on SW480 cell morphology, proliferation, and invasiveness.

SW480 KIAA1199-V5 cells were compared with SW480 Dest-V5 cells (empty vector controls). Each cell phenotype shown in this figure is based on the results of at least three independent experiments. Mean values ± SEM are reported. A. Morphology of control (left) and KIAA1199-V5-expressing cells (right) at low (top) and high (bottom) confluence. Control SW480 Dest cells, like the parental SW480 line , , tended to be round (arrow), and confluent cultures were characterized by clusters of piled-up cells (ellipse). In contrast, KIAA1199-V5-expressing cells were flatter with a more epithelial-like aspect (arrowhead). Compared with controls, KIAA1199-V5-expressing cells also displayed: B. diminished proliferation rates; C. reduced colony formation; and D. a decrease in invasiveness of ∼45%.