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First published online 25 July 2006
doi: 10.1242/jcs.03065

Journal of Cell Science 119, 3296-3305 (2006)
Published by The Company of Biologists 2006
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Efficient ADAM22 surface expression is mediated by phosphorylation-dependent interaction with 14-3-3 protein family members

Nathan J. Gödde1, Giovanna M. D'Abaco2, Lucy Paradiso1 and Ulrike Novak1,*

1 Department of Surgery, University of Melbourne, Parkville 3050, Australia
2 Department of Surgery, Royal Melbourne Hospital, Parkville 3050, Australia


Figure 1
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  		Fig. 1. Schematic of ADAM22 variant 1 full length protein and cytoplasmic tail. The cytoplasmic tail has two RSNS 14-3-3 protein consensus binding sites (black boxes in sequence below). Both 14-3-3 binding sites overlie or are adjacent to RXR motifs (underlined in sequence). C-termini are shown of variant 4 (bullet) and variant 1 as well as of the {Delta} C-terminal mutant ({blacktriangleup}).

 

Figure 2
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  		Fig. 2. Interaction between ADAM22 and three 14-3-3 protein family members in yeast two-hybrid reporter plate assays. MaV203 yeast strain containing ADAM22v1 cytoplasmic domain, ADAM22v4 cytoplasmic domain or empty pDBLeu bait vector with pPC86 library clones expressing 14-3-3 protein family members ß, {zeta}, {tau} or vector alone as isolated from a yeast two-hybrid screen. Control A: no interaction strength; control B: weak interaction strength; control C: moderately strong interaction strength; control D: strong interaction strength; control E: very strong interaction strength. Growth on SD/-Leu/-Trp master plates for bait/library plasmid maintenance, SD/-Leu/-Trp/-His/+30 mM3AT for HIS3 reporter expression, SD/-Leu/-Trp/-Ura for URA3 reporter expression, and X-gal assays on nitrocellulose membrane for lacZ reporter expression.

 

Figure 3
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  		Fig. 3. ADAM22 interacts with all 14-3-3 protein family members expressed in the brain. HEK 293T cells co-expressing FLAG-tagged full length ADAM22v4 and Myc-tagged full length 14-3-3 proteins ß, {gamma}, {epsilon}, {zeta}, {eta} or HA-tagged 14-3-3{tau} were lysed and proteins were immunoprecipitated (IP) with anti-Myc or anti-HA antibodies and probed with anti-FLAG antibody. Immunoblots were then re-probed with anti-Myc or anti-HA antibodies.

 

Figure 4
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  		Fig. 4. 14-3-3 proteins bind to ADAM22 at tandem consensus binding sites with differing affinity. (A) Immunoprecipitation (IP) from HEK 293T cell lysates co-expressing Myc-tagged full length 14-3-3ß and FLAG-tagged full length ADAM22v4 wild type, ADAM22v4 substitution mutants of the first and/or second 14-3-3 consensus binding motifs or the ADAM22v4 C-terminal deletion mutant. Proteins were immunoprecipitated with anti-Myc antibodies and immunoblots probed with anti-FLAG. As controls, immunoblots were re-probed with anti-Myc antibodies and the HEK 293T cell lysates expressing the FLAG-tagged ADAM22v4 wild-type and mutant proteins were immunoblotted and probed with anti-FLAG. (B) Purified GST-14-3-3{zeta} fusion proteins bound to glutathione-Sepharose were incubated with HEK 293T cell lysates expressing FLAG-tagged full length ADAM22v4 wild-type, ADAM22v4 substitution mutants of the first and/or second 14-3-3 consensus binding motifs or ADAM22v4 C-terminal deletion mutant. Immunoblots from subsequent GST pull-down assays were probed with anti-FLAG antibodies and then re-probed with anti-GST antibodies as control for GST-14-3-3{zeta} fusion protein levels. The HEK 293T cell lysates expressing the FLAG-tagged ADAM22v4 wild-type and mutant proteins were immunoblotted and probed with anti-FLAG as control for ADAM22 precursor and mature form protein levels. (C) MaV203 yeast strain containing pDBLeu constructs for ADAM22v1 cytoplasmic domain or ADAM22v4 cytoplasmic domain interacting with a pPC86 clone expressing 14-3-3ß and yeast containing a pDBLeu construct for the ADAM22v4 substitution mutant of the first and second 14-3-3 consensus binding motifs which no longer interacts with pPC86 library clones expressing 14-3-3 protein family members ß, {zeta} and {tau}. Growth on SD/-Leu/-Trp master plates for bait/library plasmid maintenance, SD/-Leu/-Trp/-His/+30 mM3AT for HIS3 reporter expression, SD/-Leu/-Trp/-Ura for URA3 reporter expression; and X-gal assays on nitrocellulose membrane for lacZ reporter expression.

 

Figure 5
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  		Fig. 5. The interaction between ADAM22 and 14-3-3 isoforms is dependent on the phosphorylation of ADAM22 but not the phosphorylation of 14-3-3 proteins. (A) Lysates of HEK 293T cells expressing either full-length FLAG-tagged ADAM22 or Myc-tagged or HA-tagged 14-3-3 isoforms were treated with alkaline phosphatase (ap) as indicated. Alkaline phosphatase-treated samples were subsequently treated with sodium vanadate prior to incubation with the corresponding cell lysates. Pooled lysates were then immunoprecipitated with anti-Myc-tag or anti-HA-tag antibodies and immunoblots probed with anti-FLAG antibody to detect ADAM22 precursor protein. Immunoblots were re-probed with anti-Myc-tag or anti-HA-tag antibodies as control for 14-3-3 protein expression. (B) Lysates of HEK 293T cells expressing full length FLAG-tagged ADAM22 were immunoblotted with anti-FLAG antibody as control for protein expression levels following alkaline phosphatase treatment. (C) Lysates of HEK 293T cells expressing full length FLAG-tagged ADAM22 were immunoblotted with anti-phosphoserine (pSer) antibody and re-probed with anti-FLAG antibody.

 

Figure 6
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  		Fig. 6. Stability of ADAM22 variant 4 is altered by loss of 14-3-3 binding sites. (A) HEK 293T cells expressing FLAG-tagged ADAM22v4 wild-type or a double substitution mutant of both 14-3-3 consensus binding sites were pre-treated with puromycin to block protein synthesis for 0, 2, 4, 6, or 8 hours, lysed and analysed by western blotting for FLAG-tagged ADAM22 protein levels. This experiment is representative of several previous experiments which yielded similar results. (B) Densitometry of ADAM22 precursor bands from immunoblot normalized to ß-tubulin protein levels and expressed as percentage protein levels of that of wild-type control. (C) Detection of endogenous 14-3-3 isoforms present in D645 and HEK 293T cells. RT-PCR reactions amplifying full length transcripts for 14-3-3 isoforms present in D645 and HEK 293T cDNA. NTC, no template control for 14-3-3{eta} primer set.

 

Figure 7
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  		Fig. 7. ADAM22 variant 4 mutants lacking the ability to bind 14-3-3 proteins no longer accumulate efficiently at the membrane if RXR retention motifs are still present but co-localize with Golgi markers. Immunofluorescence of D645 glioma cells transiently transfected with (A,G) FLAG-tagged ADAM22 wild type, (B,H) FLAG-tagged ADAM22 substitution mutant lacking the ability to bind 14-3-3 proteins or (C,I) FLAG-tagged ADAM22 C-terminal deletion mutant lacking both 14-3-3 binding sites and di-arginine retention motifs. (D-F, J-L) The same cells as the panels above stained with FLAG (green), DAPI (blue), and either the Golgi markers p230 (red, D,E,F) or GM130 (red, J,K,L).

 

Figure 8
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  		Fig. 8. ADAM22 variant 4 mutants lacking the ability to bind 14-3-3 proteins no longer accumulate efficiently at the membrane if RXR retention motifs are still present but co-localize with ER markers. Immunofluorescence of D645 glioma cells transiently transfected with (A,G) FLAG-tagged ADAM22 wild type, (B,H) FLAG-tagged ADAM22 substitution mutant lacking the ability to bind 14-3-3 proteins or (C,I) FLAG-tagged ADAM22 C-terminal deletion mutant lacking both 14-3-3 binding sites and di-arginine retention motifs. (D-F, J-L) The same cells as the panels above stained with FLAG (green), DAPI (blue), and either the ER markers calnexin (red, D,E,F) or calreticulin (red, J,K,L).

 

Figure 9
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  		Fig. 9. The ADAM22 variant 4 mutant lacking the ability to bind 14-3-3 proteins transports less efficiently to the cell surface. Cell surface biotinylation of D645 cells transfected with FLAG-tagged ADAM22 wild type, FLAG-tagged ADAM22 substitution mutant of the first and second 14-3-3 consensus binding motifs or FLAG-tagged ADAM22 C-terminal deletion mutant lacking both 14-3-3 binding sites and di-arginine retention motifs. Streptavidin-isolated ADAM22 proteins were detected with anti-FLAG antibody. Cell lysates were immunoblotted with anti-FLAG as control for protein expression.

 




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