Volume 8 issue 9 September 2007
Nuclear localization of endogenous RGK proteins and modulation of cell shape remodeling by regulated nuclear transport
RN Mahalakshmi, MY Ng, K Guo, Z Qi, W Hunziker and P Béguin

Supplemental Figure 1:Phosphorylation of a serine upstream from the 14-3-3 binding site regulates 14-3-3 association and modulates the subcellular distribution of Kir/Gem. (A) Preventing or mimicking phosphorylation of critical serine residues (see Fig. 5A) affects the subcellular distribution of Kir/Gem and the 14-3-3 induced nuclear clearance of Kir/Gem. COS-1 cells were transfected with cDNAs for myc-Kir/Gem in their WT form or with the indicated mutations, either alone (a-l) or with GST-14-3-3 (a’-l’ and a”-l”). Cells were then processed for immunofluorescence microscopy using Myc- or GST antibodies to label Kir/Gem (a-l and a’-l’, red) or 14-3-3 (a”-l”, green). (B) Binding of Kir/Gem mutants carrying critical serine substitutions to 14-3-3 to. (a) Precipitation experiment. GST-14-3-3 was precipitated and tested for interaction with WT or mutated Myc-Kir/Gem present in homogenates of cotransfected cells. Bound Kir/Gem was revealed by Western blot using Myc antibody. (b) Cell lysates were blotted with a Myc antibody to monitor Kir/Gem protein expression levels. As observed previously (10, 14), mutants lacking the 14-3-3 binding site displayed reduced cellular expression levels. (c) GST-14-3-3 expression levels were determined by blotting lysates with a GST antibody. (C) Quantification of the subcellular distribution of WT or mutated Kir/Gem. The subcellular distribution (none, partial or complete exclusion from the nucleus) for Myc-Kir/Gem in its WT or mutated forms was determined in 100-150 randomly chosen cells coexpressing the RGK protein and 14-3-3 in 3-5 independent experiments. This data shows that phosphorylation of S286 prevents binding of 14-3-3 to Kir/Gem and thereby affects the 14-3-3 mediated cytosolic retention of the GTP binding protein.

Figure 1 (A, B) (.jpg)
Figure 2 (C) (.jpg)

Supplemental Figure 2:C₆-ceramide induced cell shrinkage correlates with the subcellular distribution of RGK proteins. (A) Subcellular distribution of Rad, Rem and Rem2, induction of dendrite like extensions and C₆-ceramide mediated cell shrinkage (merged panels for Fig. 7A and B). Control or C₆-ceramide treated COS-1 cells transfected with cDNAs for Myc-WT Rad (panels a, b), Rad S727A (panels a’, b’) or Rad L281G (panels a”, b”), WT Rem (panels c, d), Rem S262A (panels c’, d’) or Rem L271G (panels c”, e”), or WT Rem2 (panels e, f), Rem2 S308A (panels e’, f’) or Rem2 L317G (panels e”, f”) were fixed, permeabilized and stained with anti-Myc antibodies and Hoechst to detect the RGK proteins (red) and nuclei (blue), respectively. (B) Subcellular distribution of Kir/Gem, induction of dendrite like extensions and C₆-ceramide mediated cell shrinkage. Control or C₆-ceramide treated COS-1 cells transfected with cDNAs for Myc-WT Kir/Gem (panels a-a” and d-d”), Kir/Gem S260A (panels b-b” and e-e”) or Kir/Gem W269G (panels c-c” and f-f”) were fixed, permeabilized and stained with anti-Myc antibodies and Hoechst to detect Kir/Gem (red) and nuclei (blue), respectively. White arrows indicate the nuclei of cells expressing the indicated RGK protein. (C) Quantification. The ability of C₆-ceramide to induce cell shrinkage (as evidenced by the compression of the cell body and nucleus) was monitored in 100 randomly chosen cells expressing the indicated WT or mutated Kir/Gem proteins. (D) C₆-ceramide induced cell shrinkage depends on the presence of RGK proteins. Control (panels a-a”) or C₆-ceramide treated (panels b-b”) COS-1 cells transfected with an N-terminally HA-tagged β-gal cDNA were fixed, permeabilized and stained with anti-HA antibodies and Hoechst to detect β-gal (green) and nuclei (blue), respectively. White arrows indicate the nuclei of cells expressing the indicated RGK protein.

Figure 3 (A, B, C, D) (.jpg)