FBP17b, FBP17, and CIP4 interact with AKAP350. (A) Genomic organization of human FBP17. The 15 exons are denoted, with the dashed boxes indicating the exons that are spliced out in the FBP17B transcript. The splicing does not interrupt the exons comprising the MBD, the cdc42 interacting domain (CID), or the SH3 domain. (B) Diagram showing identity (top numbers) and similarity (bottom numbers) between FBP17 exons 1-15, and CIP4 exons 1-14. (C) FBP17, FBP17b, CIP4, and the CIP4 deletion constructs of the MBD or the C-terminal SH3 domain were cloned in pBD-Gal4 vector. The interaction of these proteins with AKAP350 was assessed in yeast two-hybrid binaries assays with pAD-AKAP350(1076-2143). The results confirm FBP17b interaction and indicate that FBP17 and CIP4 also interact with AKAP350(1076-2143) and that the MBD, but not the SH3 domain, in CIP4 is necessary for the interaction. (D) In vitro confirmation of AKAP350/CIP4 interaction. (His)₆-tagged CIP4 was attached to Ni beads. A 100,000 × g gastric mucosal supernatant (SM) was incubated with His-CIP4 Ni beads (CIP4) or Ni beads alone (blank). After incubation, samples were washed and eluted with sample buffer. Samples were subjected to electrophoresis in 3-10% SDS-PAGE followed by Western blotting with anti-AKAP350 monoclonal antibody (14G2). Bar, 5 μm.

CIP4 colocalizes with AKAP350 at the Golgi. (A) Cellular extracts from MDCK cells were analyzed for immunoreactivity with the rabbit anti-CIP4 (r-CIP4) antibody and compared with the monoclonal anti-CIP4 antibody. The Western blot of r-CIP4 immunoprecipitate (IP), and the 100,000 × g microsomal pellet (P100) and supernatant (SN) detect a main band with an apparent molecular mass of approximately 75 kDa (a). We also observed a higher band with weaker immunoreactivity at 83 kDa (b). Both antibodies recognized the same bands. The lower mass band in the immunoprecipitated (IP) lane blotted with r-CIP4 corresponds to the heavy chain of the antibody used in the immunoprecipitation. (B-E) MDCK cells were fixed and dual stained with r-CIP4 (B and D) and anti-AKAP350 (14G2) (C) or antibody against the Golgi apparatus marker p58 (E). The immunofluorescence images show extensive areas of colocalization of CIP4 and AKAP350 (upper row) in Golgi elements. The arrow indicates centrosomal staining of AKAP350 that did not show any CIP4 immunoreactivity. (F-K) MDCK cells were treated with 15 μM nocodazole for 1 h, fixed, and dual-stained with r-CIP4 (H and J) and anti-AKAP350 (I) or anti-p58 (K) antibodies. F and G show α-tubulin staining in control or nocodazole-treated cells respectively. (L-Q) MDCK cells were treated with 5 μM BFA for different periods. The figures show the dual staining with r-CIP4 (L and N) and anti-cdc42 (M and O) antibodies of control cells (L and M) or cells treated with BFA for 1 min (N and O) and cells treated for 1 h with BFA stained with r-CIP4 (P) and 14G2 anti-AKAP350 (Q) antibodies. Bar, 5 μm.

CIP4 is phosphorylated by PKA. (A) In vitro phosphorylation. (His)₆-tagged CIP4 attached to Ni beads was incubated with the catalytic subunit of PKA in the appropriate buffer at 30°C for 10 min. The experiment was also performed in the presence of the PKA inhibitor H-89 (1 and 10 μM). As controls, we incubated Ni beads without bound protein with PKA (first lane) and (His)₆-CIP4-Ni beads without PKA (second lane). The reactions were terminated by heating the samples in sample buffer at 65°C for 20 min. The samples were resolved by SDS-PAGE and transferred to Immobilon membranes, and CIP4 phosphorylation was analyzed using an antibody that recognizes the RXXS/T(Pi) site. (B and C) In situ analysis of CIP4 phosphorylation. MDCK cells were grown to confluence and stimulated with different doses of forskolin (F) for 10 min (B), or alternatively were preincubated for 20 min in the presence or absence of 10 μM myristoylated PKI and then stimulated with 5 μM forskolin for 3 min (C). After the incubations, cells were scraped and lysed in the presence of protease and phosphatase inhibitors, and CIP4 immunoprecipitates were prepared as described and analyzed for phosphorylation as in A. (A-C) Representative of three separate experiments. For quantification of phosphorylated CIP4, we performed the densitometry of the bands using the NIH Image J program and divided the density of each RXXT/S(Pi) immunoreactive band by that of the same band with the monoclonal anti-CIP4 antibody. The analysis of the in situ phosphorylation indicated that forskolin induced an increase on CIP4 phosphorylation in the RXXS/T site of 88%, and this increase was prevented by preincubation with the PKA inhibitor.

AKAP350(1076-2143) expression alters the structure of the Golgi apparatus. MDCK cells were transiently transfected with GFP-AKAP350(1076-2143) and grown for 20 h. Fixed cells were dual stained with r-CIP4 and anti-AKAP350 or anti-p58 antibodies (A), or with anti-giantin and anti-golgin-97 or anti-α-tubulin antibodies (B). The viability of the cells was confirmed by analysis of the DAPI staining and the phase contrast image of the cells (our unpublished data). The stars indicate the positions of cells expressing AKAP350(1076-2143)-GFP. Bar, 5 μm.

siRNA1 and siRNA2 induce a decrease in AKAP350 expression. HeLa cells were transfected with RNA duplexes targeted to two different AKAP350 specific sequences (siRNA1 and siRNA2) or with the RNA duplexes synthesized using a scrambled sequence of the siRNA1 target sequence (siRNA control) and allowed to grow for 48 h. The cells were scraped and lysed in the presence of protease inhibitors. The samples were resolved by SDS-PAGE and transferred to nitrocellulose or Immobilon membranes for the analysis of AKAP350 or CIP4 expression. The densitometry of the bands indicated a reduction of total AKAP350 protein to 20% of control with the siRNA1 and to 40% of control with the siRNA2 transfected cells. The RNA interference for AKAP350 expression did not modify the CIP4 protein levels.

Reduction of AKAP350A expression by RNA interference alters CIP4 distribution and induces vesiculation of the Golgi apparatus. HeLa cells were transfected with the AKAP350A-targeted RNA duplexes siRNA1 and siRNA2 or with the siRNA control, and allowed to grow for 72 h. After fixation, cells were dual stained with the 14G2 anti-AKAP350 and r-CIP4 antibodies (first column), anti-AKAP350A and anti-p58 (second column), anti-AKAP350A and anti-golgin-97 (third column), and anti-AKAP350A and anti-α-tubulin (fourth column). Approximately 90% of the cells transfected with siRNA1 and siRNA2 showed a decrease in AKAP350A staining that was more prominent in the cells expressing the siRNA1. Approximately 40% of the siRNA1 and 20% of the siRNA2 cells showed no AKAP350A staining. The reduction in AKAP350 expression was confirmed by Western blot (see Figure 5). The viability of the cells was confirmed by evaluation of the DAPI staining and the phase contrast images of the cells (our unpublished data). Bar, 2.5 μm.