JCS02670 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Effect of expression levels of MARK4 on purification of interacting proteins. 293 cells were transiently transfected with the same pEGFP-C2-TAP-MARK4 expression vector used to generate stable cell lines in Fig. 1 and lysed 36 hours post-transfection. In parallel, 293 cell lines stably expressing a low level and a 5-10-fold higher level of GFP-TAP-MARK4 were cultured and lysed (upper panel). 10 mg of cell extract was immunoblotted with an antibody recognizing GFP (lower panel). The TAP-tagged MARK4 from each of the cell lysates was affinity purified and electrophoresed on a polyacrylamide gel and the protein bands visualized following colloidal Coomassie Blue staining. The bands that we identified by mass spectrometry are indicated. Bands that were not reliably identified are left unlabelled.

• Supplemental Figure 2 -

  Fig. S2. Analysis of phosphorylation of TORC2. GST-SIK (A) and GST-QSK (B) either complexed to 14-3-3 (+14-3-3 black line) or dissociated from 14-3-3 (–14-3-3 red line) was generated as described in the legend to Fig. 4. GST-TORC2 was phosphorylated by these preparations of QSK and SIK for 10 minutes under conditions in which phosphorylation was in the linear rate (data not shown). The ³²P-labelled TORC2 was isolated by electrophoresis on a polyacrylamide gel and stoichiometry of phosphorylation that was obtained under each condition is indicated. The ³²P-labelled TORC2 was digested with trypsin and the resulting peptides were chromatographed on a C18 column. Fractions containing the major ³²P-labelled peptides are marked. (C) The indicated peptides were analysed by MALDI TOF–TOF mass spectrometry and the mass (M) of peptides are calculated from m/z observed. The site of phosphorylation within each peptide was determined by solid phase Edman sequencing in which ³²P-radioactivity was measured after each cycle of Edman degradation. The cycle number in which ³²P-radioactivity was released is indicated. The deduced amino acid sequences of P₁, P₂, P₃, P₄ and P₅ are indicated in which the phosphorylated Ser residues are underlined. P₆ did not couple to the arylamine membrane employed for solid phase Edman sequencing, and we were unable to locate the phosphorylation site within this peptide.