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First published online 9 August 2005
doi: 10.1242/jcs.02496

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PCTAIRE protein kinases interact directly with the COPII complex and modulate secretory cargo transport

Department of Biochemistry, University of Bristol, School of Medical Science, University Walk, Bristol, BS8 1TD, UK

View larger version (24K): [in a new window]   Fig. 1. Interaction of Sec23Ap with PCTAIRE protein kinases. Yeast two-hybrid screening of a human brain cDNA library with full-length Sec23Ap identified a clone encoding apart of PCTAIRE-3b. (A) Interactions were reconfirmed by transformation and shown to occur specifically with Sec23Ap. A weak association was also seen with Sec24Dp but not with lamin or Sec13p. The weakly detectable interaction with Sec31Ap in plate growth assays was not seen in complementary colorimetric assays and therefore was deemed a false positive. (B) The two-hybrid clone encoding PCTAIRE-3 includes nine amino acids from predicted intron c and 110 amino acids from predicted intron h. Using PCR, we generated a `fully-spliced' cDNA including exons 4-8, which is equivalent to the central region of the predominant isoform of PCTAIRE-3, PCTAIRE-3a (Herskovits and Davies, 2004; Okuda et al., 1992). The amino acids encoded by predicted intron h are believed to arise from incomplete splicing as they include a stop codon after 61 amino acids. The nine amino acids included in this clone would encode a known splice form, PCTAIRE-3b (Herskovits and Davies, 2004). (C) Further yeast two-hybrid assays showed that Sec23Ap and Sec24Dp also interact with PCTAIRE-3a and PCTAIRE-1. DDO, double dropout medium; QDO, quadruple dropout medium. (D) Specificity of the two-hybrid interaction was determined using the PSTAIRE kinase CDK2. Co-transformants were assessed for interaction between CDK2 and lamin, Sec23Ap, Sec24Dp, Sec13p and Sec31p (upper panels). No interactions were seen. Concomitant transformation of yeast with Sec23Ap and the PCTAIRE-3 clone isolated from the library screen was used as a positive control (lower panels).

View larger version (30K): [in a new window]   Fig. 2. PCTAIRE-1 and Sec23Ap interact directly in vitro. Purified recombinant FLAG-Sec23Ap was incubated with immobilized GST-fusion proteins as follows: GST, GST-PCTAIRE-1, GST-PCTAIRE-1(K194R) or GST-PCTAIRE-1(S153A). Beads were washed and bound protein eluted with sample buffer containing SDS, separated by SDS-PAGE and immunoblotted with an anti-FLAG antibody. Right-hand blot contains 5% of the amount of FLAG-Sec23Ap included in the binding assays (input). Position of molecular mass markers in kDa is indicated on the left.

View larger version (31K): [in a new window]   Fig. 3. PCTAIRE kinases are expressed in many cell lines and can be co-immunoprecipitated with Sec23Ap. (A) Analysis of PCTAIRE expression in cell lysates from four different cell lines: HeLa (cervical epithelia), A549 (lung epithelia), HEK-293 (embryonic kidney), MDCK (Madin-Darby canine kidney). (B) Sec23Ap can be co-immunoprecipitated with PCTAIRE-3 from MDCK cell lysates (lanes 1-3) and PCTAIRE-1 from HeLa cell lysates (lanes 4-6). Immunoprecipitates were washed extensively, separated by SDS-PAGE followed by immunoblotting with anti-Sec23p. Lanes 1 and 4, 5% input lysate; lanes 2 and 5, immunoprecipitation with anti-PCTAIRE-3 (lane 2) or anti-PCTAIRE-1 (lane 5); lanes 3 and 6, control immunoprecipitation using rabbit IgG. *, non-specific band at 100 kDa. (C) Specificity of co-immunoprecipitation was confirmed using antibodies directed against CDK1. Unlike anti-PCTAIRE-1, neither mouse IgG nor anti-CDK1 was able to immunoprecipitate Sec23Ap from HeLa cell lysates. (D) In the converse experiments, neither anti-Sec23Ap nor anti-PCTAIRE-1 were able to immunoprecipitate CDK1.

View larger version (89K): [in a new window]   Fig. 4. Depletion of PCTAIRE-1 expression using RNAi causes disruption of morphology of the early secretory pathway. HeLa cells were transfected with siRNA duplexes targeting lamin or PCTAIRE-1. After 72 hours, cells were lysed and blotted for PCTAIRE-1 (A) or lamin (B). (C) Cells grown on coverslips, transfected with siRNA specific for PCTAIRE-1 and incubated for 72 hours. Cells were then fixed and processed for immunofluorescence with antibodies specific to GM130, ERGIC-53, Sec24Dp, giantin, -tubulin or the KDEL-receptor. (D) Enlargement of the boxed regions of ERGIC-53 immunolabelling (green) from C show that PCTAIRE-1 siRNA results in increases ER labelling clearly shown by the nuclear envelope localization. The nucleus (DNA labelled with DAPI) is shown in blue.

View larger version (54K): [in a new window]   Fig. 5. Kinase-dead PCTAIRE-1 disrupts protein localization within the early secretory pathway. Cells were transfected with plasmids to express GFP-PCTAIRE-1 (wild type or point mutants as indicated). (A) The K184R ('kinase-dead') point mutation in PCTAIRE-1 results in fragmentation of the Golgi (giantin) and redistribution of ERGIC-53 to a more dispersed localization. In contrast, the wild type or `active' mutants of PCTAIRE-3 do not cause these effects. (B) The K184R point mutation in PCTAIRE-1 results in redistribution of the ER-Golgi SNARE protein membrin to a more dispersed localization (asterisk), and a reduction in the intensity and juxtanuclear clustering of COPII-coated ERES marked with Sec24Dp. In contrast, the wild type or `active' mutants of PCTAIRE-3 do not cause these effects.

View larger version (18K): [in a new window]   Fig. 6. Expression of kinase-dead PCTAIRE-1 causes an inhibition of secretory cargo transport. Quantitative transport assays using ts045-G-CFP was performed using cells transfected with YFP, kinase-dead mutant YFP-PCTAIRE-1(K194R), or active mutant YFP-PCTAIRE-1(S153A). Histogram of the mean amount of ts045-G-CFP transported to the plasma membrane after a 60-minute incubation at 32°C. The points indicate the scatter of values from ten cells for each experiment. Errors (ANOVA) show statistical significance for the inhibition of transport following PCTAIRE-1(K194R) expression.