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. 2019 Feb 17;18(3):e12906. doi: 10.1111/acel.12906

Figure 5.

Figure 5

KIN‐4 physically interacts with DAF‐18/PTEN. (a‐c) Co‐localization of KIN‐4a::GFP and mCherry::DAF‐18. (a) DAF‐18 was mainly expressed in several head neurons (arrowhead). Arrows indicate red fluorescent signals of injection marker. Scale bar is 200 μm. (b) Shown in a blue rectangle is an enlarged picture of the head part in panel A. (c) Deconvolution images of a neuron. Subcellular localization of KIN‐4a::GFP and mCherry::DAF‐18 in a head neuron of mCherry::daf‐18; kin‐4a::gfp; ofm‐1p::rfp animals. Scale bar is 10 μm. (d) The PDZ domain of KIN‐4 (green) overlaps with that of MAST2 (magenta), one of human MAST family kinases. (e) The marked resides (V1186, V1188, I1202, and L1257) are conserved in the predicted structure of the PDZ domain of KIN‐4 (KIN‐4 PDZ, green) with that of human MAST2. F951 is a conserved residue in the C‐terminal region of DAF‐18 (DAF‐18 C, purple, See Supporting information Figure S8). DAF‐18 C from the 951st to the 962nd residues was predicted by substituting each residue of PTEN (2KYL) with a Coot program. (f) HA‐DAF‐18 was co‐immunoprecipitated by immunoprecipitating FLAG‐KIN‐4. IP: immunoprecipitation. IB: immunoblotting (g) A GST pull‐down assay of HA tag‐fused DAF‐18 [HA‐DAF‐18] with GST‐fused KIN‐4 fragment proteins. HA‐DAF‐18 was pulled down by using a PDZ domain‐containing KIN‐4 C‐terminal fragment [C], but not by an N‐terminal KIN‐4 fragment [N] or by a kinase domain‐containing KIN‐4 middle fragment [Mid]. See the upper illustration that depicts the domain regions in KIN‐4. (h) Illustration of wild‐type and deletion mutant DAF‐18 that were used for GST pull‐down assays. Wild‐type DAF‐18 [WT] has the intact DAF‐18 C‐terminal PDZ‐binding motif. DAF‐18 deletion mutant proteins, [Δ4C] and [Δ12C], do not contain the last 4 and 12 amino acids from its C‐terminal ends, respectively. (i) GST pull‐down assays using wild‐type or mutant FLAG‐tagged full‐length DAF‐18 [FLAG‐DAF‐18] with GST‐fused KIN‐4 PDZ domain [GST‐KIN‐4 PDZ] proteins. GST KIN‐4 PDZ strongly bound to FLAG‐DAF‐18 WT [WT] but weakly to FLAG‐DAF‐18 Δ4C [Δ4C] and FLAG‐DAF‐18 Δ12C [Δ12C]