File S1 - Direct Interaction between AR and PAK6 in Androgen-Stimulated PAK6 Activation

Supporting Figures S1-S6. Figure S1. Identification of Serine-308 as a site of phosphorylation. The amino acid sequence is provided above the spectrum, and the masses above and below the sequence correspond to the theoretical b- and y-type product ions, respectively. The masses provided are the singly-protonated, monoisotopic product ion masses. The observed singly-protonated product ions are underlined. For simplicity, all doubly protonated ions are not labeled in the spectra as they exist at 50% abundance or less. Asterisks indicate ions that result from neutral loss of H3PO4. Figure S2. Identification of Threonine-326 as a site of phosphorylation. The amino acid sequence is provided above the spectrum, and the masses above and below the sequence correspond to the theoretical b- and y-type product ions, respectively. The masses provided are the singly-protonated, monoisotopic product ion masses. The observed singly-protonated product ions are underlined. Asterisks indicate ions that result from neutral loss of H3PO4 from fragment ions. Additionally, the presence of the second site of phosphorylation is determined due to accurate mass of the peptide, however, the first fragment ion representing this site is located at y7, thus the phosphorylation could exist in any of the sites shown in brackets. Figure S3. Identification of Serine-346 as a site of phosphorylation. The amino acid sequence is provided above the spectrum, and the masses above and below the sequence correspond to the theoretical b- and y-type product ions, respectively. The masses provided are the singly-protonated, monoisotopic product ion masses. The observed singly-protonated product ions are underlined. The site of phosphorylation cannot be definetively identified, due to lack of specific ions related to either site of phosphorylation, however we hypothesize that the phosphorylation is on the first of the serine residues due to lack of tryptic cleavage at the preceding site. Asterisks indicate ions that result from neutral loss of H3PO4. Figure S4. Identification of Thrionine-354 and Serine-360 as sites of phosphorylation. The amino acid sequence is provided above the spectrum, and the masses above and below the sequence correspond to the theoretical b- and y-type product ions, respectively. The masses provided are the singly-protonated, monoisotopic product ion masses. The observed singly-protonated product ions are underlined. Asterisks indicate ions that result from neutral loss of H3PO4 from fragment ions. This doubly phosphorylated peptide loses two phosphoric acid groups from the parent ion which are marked with *. Figure S5. Identification of Serine-360 as a site of phosphorylation. The amino acid sequence is provided above the spectrum, and the masses above and below the sequence correspond to the theoretical b- and y-type product ions, respectively. The masses provided are the singly-protonated, monoisotopic product ion masses. The observed singly-protonated product ions are underlined. Asterisks indicate ions that result from neutral loss of H3PO4 from fragment ions. Figure S6. Identification of Serine560 as a site of phosphorylation. The amino acid sequence is provided above the spectrum, and the masses above and below the sequence correspond to the theoretical b- and y-type product ions, respectively. The masses provided are the singly-protonated, monoisotopic product ion masses. The observed singly-protonated product ions are underlined. Asterisks indicate ions that result from neutral loss of H3PO4. (PDF)