JCS02313 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. (A) 293T lysates containing exogenously expressed HA-tagged wild-type GEF-H1 and the phosphorylation point mutant GEF-H1 S810A was co-expressed with PAK4 kinase domain. The upper panel shows expression of the HA-tagged proteins detected using a HA monoclonal and the lower shows detection with the phosphospecific GEF-H1 antiserum. (B) Filters assayed in a dot blot were spotted with the indicated amounts of peptide and probed with phosphospecific GEF-H1 antiserum. Two dilutions are shown.

• Supplemental Figure 2 -

  Fig. S2. (A) PAK4 phage display hits (above the broken line) reduced to consensus target sequences by alignment to a PAK4 substrate consensus. Asterisk indicates the phospho-acceptor site. An N-terminal GEF-H1 peptide (below the broken line) with a predicted phosphorylation site is marked with an asterisk. (B) Peptide phospho-isomers derived from potential regulatory regions used to confirm phosphorylation sites. Residues printed in red are pre-phosphorylated and block phosphorylation in vitro. (C) A combination of deletion and site-directed mutants used to physically located PAK4 phosphorylation on the GEF-H1 protein.

• Supplemental Figure 3 -

  Fig. S3. Conserved residues with Cdc24 are shaded in pink and yellow. PAK4 phosphorylation sites are highlighted by asterisks on the weighted consensus, which is aligned to the putative regulatory regions conserved with Cdc24.

• Supplemental Figure 4 -

  Fig. S4. All experiments were controlled by monitoring the effects of co-expressing PAK4 with the EGFP vector alone (I-III). PAK4 proteins are detected by staining with anti-HA followed by immunofluorescence labeling with anti-mouse rhodamine conjugate PAK4 and EGFP protein is green fluorescent. PAK4 wild-type together with EGFP do not produce any notable actin structures (I). Activated PAK4 together with EGFP seems to cause cell enlargement (note scale bar) compared with non-transfected cells, consistent with Rho inhibition (II). Cytoplasmic aggregates of kinase-dead PAK4 K350,351A are seen when overexpressed in NIH-3T3 cells (III).

• Supplemental Figure 5 -

  Fig. S5. NIH-3T3 cells expressing GEF-H1S, the mutants GEF-H1S S810A, GEF-H1 S67A and GEF-H1S S67A,S810A together with activated PAK4 S474E were examined for MTs by fluorescence imaging of EGFP for GEF-H1, PAK4 antiserum followed with anti-rabbit fluorescent rhodamine for PAK4, and anti-b-tubulin followed by anti-mouse Marina Blue conjugate. GEF-H1S and the mutant GEF-H1S S810A causes dissolution and depolarization of b-tubulin structures (top two rows) with less intense staining of MTs. MTs appear stabilized in the presence of the mutant proteins GEF-H1S S67A and GEF-H1S S67A,S810A.