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Functional interaction between the SSeCKS scaffolding protein and the cytoplasmic domain of ß1,4-galactosyltransferase

Michael J. Wassler^1,*, Cynthia I. Foote^1,*, Irwin H. Gelman^2,* and Barry D. Shur^1,

¹ Department of Cell Biology, Emory University School of Medicine, Room 100, 1648 Pierce Drive, Atlanta, GA 30322, USA
² Department of Microbiology, Mount Sinai School of Medicine, Box 1124, One Gustave L. Levy Place, New York, NY 10029, USA
^* These authors contributed equally to this work

View larger version (11K): [in a new window]   Fig. 1. Two clones, 1.12 and 2.52, show specificity for the GalT cytoplasmic domain in the two-hybrid system. Relative ß-galactosidase activity, normalized to cell number, using the MUG assay as described in Materials and Methods. The interaction of Ras/Raf was used as a positive control and the level of background activity between 1.12, 2.52 and Ras was used as a negative control. The error bars represent the standard error from eight experiments.

View larger version (89K): [in a new window]   Fig. 2. 1.12 and 2.52 show homology to two independent regions of mouse SSeCKS. Amino acid sequence of mouse SSeCKS is shown (GenBank accession no. AB020886). Clone 1.12 corresponds to amino acids 51-331 (bold) and 2.52 corresponds to amino acids 1278-1562 (bold).

View larger version (92K): [in a new window]   Fig. 3. GFP-1.12 and BFP-2.52 colocalize with GalT at distinct subcellular locations. Confocal microscopy of 3T3 fibroblasts stably transfected with GFP only (A-C), GFP-1.12 (D-F,G-I) or BFP-2.52 (J-L). The cells were immunostained for GFP (A,D,G,J) or GalT (B,E,H,K) using monoclonal antibodies to GFP, or polyclonal antibodies against recombinant murine GalT, respectively, as described in Materials and Methods. GFP-1.12 localizes to cell lamellipodia, whereas BFP-2.52 is confined to the perinuclear region, both of which are coincident with GalT as represented by merged yellow images (C,F,I,L). Arrowheads indicate tips of filopodia.

View larger version (11K): [in a new window]   Fig. 4. 1.12 and 2.52 coimmunoprecipitate with GalT. (A) Lysates from cells stably transfected with GFP-1.12 were immunoprecipitated (Ip) with rabbit preimmune sera (PI) or sera raised against recombinant GalT (I) in the presence or absence of calcium, magnesium or phosphatidyl serine (PS) and immunoblotted with GFP antibodies. (B) Lysates from cells stably transfected with BFP-2.52 were immunoprecipitated with preimmune sera (PI) or antibodies against GalT (I) and immunoblotted with GFP antibodies. In both A and B the electrophoretic mobility of the immunoprecipitated GFP/BFP fusion protein (arrows) was determined by immunoblotting samples of the cell lysates (L) with anti-GFP antibodies. (C) Lysates from GFP-1.12 and BFP-2.52 expressing cells were immunoprecipitated with either control sera (PI) or antibodies against GFP (I), resolved under non-reducing conditions, and immunoblotted with antibodies against GalT. Molecular weight markers are shown to the left of each panel.

View larger version (18K): [in a new window]   Fig. 5. Endogenous, full-length SSeCKS coimmunoprecipitates with GalT. NIH 3T3 cell lysates were immunoprecipitated with either preimmune sera (PI) or antibodies against recombinant GalT (I) and immunoblotted with anti-SSeCKS antibodies. The electrophoretic migration of SSeCKS in the 3T3 cell lysate (L) is shown by immunoblotting for SSeCKS. Molecular mass markers are shown to the left.

View larger version (13K): [in a new window]   Fig. 6. SSeCKS interacts with the GalT cytoplasmic domain in mouse mammary gland cells. (A) Lysates from NMuMG cells expressing GFP only (NMuMG/GFP) or GFP fused to the GalT cytoplasmic and transmembrane domain (NMuMG/TL-GFP) were immunoprecipitated (Ip) with preimmune sera (PI) or with antibodies against SSeCKS (I) and immunoblotted with monoclonal antibodies against GFP. The electrophoretic migration of the expressed GFP and TL-GFP proteins is shown by immunoprecipitation with anti-GFP. Anti-SSeCKS antibodies coprecipitate the TL-GFP fusion protein, whereas GFP does not precipitate with SSeCKS. (B) Lysates from nontransfected (NMuMG) and transfected (NMuMG/TL-GFP) cells were immunoprecipitated with preimmune sera (PI), antibodies against recombinant GalT (I), or antibodies against GFP and immunoblotted with SSeCKS antibodies. Anti-GalT antibodies readily coprecipitate SSeCKS in NMuMG parental cells. Overexpression of the GalT cytoplasmic domain (TL-GFP) leads to a consequent increase in the amount of coprecipitating SSeCKS. The electrophoretic behavior of SSeCKS in NMuMG cell lysates (L) is shown. Molecular mass markers are shown to the left.

View larger version (24K): [in a new window]   Fig. 7. SSeCKS is enriched in the insoluble fractions of cell lysates. Total cell lysate (T), soluble fraction (S) and insoluble pellet (I) were resolved by 6% PAGE and subsequently immunoblotted with polyclonal SSeCKS antibody. To verify the fractionation of soluble and insoluble fractions, parallel assays were probed with antibodies against E-cadherin.

View larger version (65K): [in a new window]   Fig. 8. Expressing the cytoplasmic and transmembrane domains of GalT (TL) creates a cell spreading defect that is reversed by co-expression of GFP-1.12. (A) nontransfected NMuMG cells. (B) NMuMG cells stably transfected with TL-GFP. (C) NMuMG cells stably transfected with both TL-GFP and GFP-1.12. (D) NMuMG cells stably transfected with both TL-GFP and empty BFP vector. Representative lysates of 1.12 and TL-GFP double transfectants (clones 3 and 8) were immunoprecipitated with anti-GFP antibodies and stained for GFP fusion proteins using polyclonal anti-GFP (E) to confirm the expression of the 1.12 fusion protein.