Table 1.
MRTF-A phosphorylation sites. NIH3T3 cells stably expressing Flag-MRTF-A were stimulated with serum for 30 min, immunoprecipitated with Flag antibody and fractionated by SDS-PAGE in triplicate. The MRTF-A bands were excised, digested with Trypsin, AspN or Chymotrypsin and the digests analysed with and without titanium dioxide enrichment on the LTQ Orbitrap Discovery. Fixed modifications were set as Carbamidomethyl (C) and variable modifications as Oxidation (M) and Phospho (STY). The estimated false discovery rate was set to 1% at the peptide, protein and site level. For all enzymatic digests, a maximum of two missed cleavages was allowed. Raw data were processed in MaxQuant (version 1.3.0.5) for peptide and protein identification, searching against the canonical sequence of Mus Musculus from the Uniprot KB, release 2012_08. The summary table was generated from the MaxQuant output file PhosphoSTY Sites.txt, an FDR-controlled site-based table compiled by MaxQuant from the relevant information about the identified peptides. Modified residues are shown in the left columns, with residue positions for MRTF-A(fl) and MRTF-A(met) indicated. In the sequence window the central residue is the putative modified site, shown in bold. For each site, the MS data are summarised as the best peptide Posterior Error Probability (PEP) score and the localisation probability, and/or the availability of a phospho-site specific antibody indicated. Blue shading indicates sites denoted reliable on the basis of their satisfying PEP <1.0E-06 and Localisation Probability >0.75 (>0.5 for repeated phosphoacceptors), confirmation by phosphosite-specific antibody, or manual peptide identification for residues 587 and 601 (peptides TQLTLQAS(ph)PL and AAS(ph)C(carb)C(carb)LS(ph)PGAR respectively, shown in blue in the sequence window). Residues mutated to Alanine in MRTF26A are shown in red. Peptides used for immunisation are underlined; those used for S98, S231, S663, S867 and ST897/9 were synthesised with C-terminal cysteines.
| Amino acid | MRTF-A(FL) | MRTF-A(Met) | Sequence | PEP score | Localizn prob. | Phospho-antibody |
|---|---|---|---|---|---|---|
|
S |
33 |
n/a |
ENDDEPVLLSLSAAPSPQSEAVANELQELSL |
1.39E-10 |
0.993037 |
pAb33 |
|
S |
98 |
6 |
TREELVSQGIMPPLKSPAAFHEQRRSLERAR |
1.16E-06 |
1 |
pAb98 |
|
S |
206 |
114 |
IIVGQVNYPKVADSSSFDEDSSDALSPEQPA |
0.0211238 |
0.310963 |
|
|
S |
211 |
119 |
VNYPKVADSSSFDEDSSDALSPEQPASHESQ |
0.000127434 |
0.799217 |
|
|
S |
212 |
120 |
NYPKVADSSSFDEDSSDALSPEQPASHESQG |
0.0059612 |
0.371831 |
|
|
S |
216 |
124 |
VADSSSFDEDSSDALSPEQPASHESQGSVPS |
1.67E-30 |
1 |
|
|
S |
231 |
139 |
SPEQPASHESQGSVPSPLESRVSDPLPSATS |
1.69E-23 |
1 |
pAb231 |
|
S |
248 |
156 |
LESRVSDPLPSATSISPTQVLSQLPMAPDPG |
4.25E-16 |
0.999792 |
pAb248 |
|
T |
402 |
310 |
APPKPSAETPGSSAPTPSRSLSTSSSPSSGT |
0.000114318 |
0.993969 |
|
|
S |
406 |
314 |
PSAETPGSSAPTPSRSLSTSSSPSSGTPGPS |
9.19E-10 |
0.559961 |
|
|
S |
408 |
316 |
AETPGSSAPTPSRSLSTSSSPSSGTPGPSGL |
2.23E-11 |
0.676126 |
|
|
T |
409 |
317 |
ETPGSSAPTPSRSLSTSSSPSSGTPGPSGLA |
2.49E-09 |
0.497067 |
|
|
S |
412 |
320 |
GSSAPTPSRSLSTSSSPSSGTPGPSGLARQS |
2.23E-11 |
0.99797 |
|
|
S |
415 |
323 |
APTPSRSLSTSSSPSSGTPGPSGLARQSSTA |
4.58E-06 |
0.524459 |
|
|
T |
417 |
325 |
TPSRSLSTSSSPSSGTPGPSGLARQSSTALA |
2.34E-09 |
0.998496 |
|
|
S |
428 |
336 |
PSSGTPGPSGLARQSSTALAAKPGALPANLD |
0.000953078 |
0.745616 |
|
|
T |
429 |
337 |
SSGTPGPSGLARQSSTALAAKPGALPANLDD |
0.000953078 |
0.493371 |
|
|
S |
480 |
388 |
KTELIERLRAYQDQVSPAPGAPKAPATTSVL |
2.99E-06 |
1 |
|
|
S |
541 |
449 |
MVVATVTSNGMVKFGSTGSTPPVSPTPSERS |
1.93E-11 |
0.96512 |
|
|
T |
542 |
450 |
VVATVTSNGMVKFGSTGSTPPVSPTPSERSL |
3.23E-07 |
0.899805 |
|
|
S |
544 |
452 |
ATVTSNGMVKFGSTGSTPPVSPTPSERSLLS |
3.78E-08 |
0.916768 |
|
|
T |
545 |
453 |
TVTSNGMVKFGSTGSTPPVSPTPSERSLLST |
2.23E-12 |
0.994425 |
|
|
S |
549 |
457 |
NGMVKFGSTGSTPPVSPTPSERSLLSTGDEN |
2.23E-12 |
0.999632 |
|
|
T |
551 |
459 |
MVKFGSTGSTPPVSPTPSERSLLSTGDENST |
2.23E-12 |
0.94872 |
|
|
S |
553 |
461 |
KFGSTGSTPPVSPTPSERSLLSTGDENSTPG |
3.78E-08 |
0.998455 |
|
|
S |
559 |
467 |
STPPVSPTPSERSLLSTGDENSTPGDAFGEM |
0.00405521 |
0.999782 |
|
|
T |
560 |
468 |
TPPVSPTPSERSLLSTGDENSTPGDAFGEMV |
0.00405521 |
0.499997 |
|
|
S |
565 |
473 |
PTPSERSLLSTGDENSTPGDAFGEMVTSPLT |
0.000480998 |
0.837039 |
|
|
S |
577 |
485 |
DENSTPGDAFGEMVTSPLTQLTLQASPLQIV |
5.33E-09 |
0.999998 |
|
|
S |
587 |
495 |
GEMVTSPLTQLTLQASPLQIVKEEGARAASC |
0.00523736 |
1 |
|
|
S |
601 |
509 |
ASPLQIVKEEGARAASCCLSPGARAELEGLD |
0.000780733 |
1 |
|
|
S |
605 |
513 |
QIVKEEGARAASCCLSPGARAELEGLDKDQM |
2.27E-13 |
1 |
pAb605 |
|
S |
662 |
570 |
LQLEQQKRAQQPAPASSPVKRESGFSSCQLS |
1.02E-08 |
0.998348 |
|
|
S |
663 |
571 |
QLEQQKRAQQPAPASSPVKRESGFSSCQLSC |
0.000148519 |
0.998905 |
pAb663 |
|
S |
708 |
616 |
VPTTNHGDTQAPAPESPPVVVKQEAGPPEPD |
1.17E-24 |
1 |
pAb708 |
|
S |
744 |
652 |
QLLLGSQGTSFLKRVSPPTLVTDSTGTHLIL |
n/a |
n/a |
pAb744 |
|
S |
775 |
683 |
TVTNKSADGPGLPAGSPQQPLSQPGSPAPGP |
7.59E-16 |
1 |
pAb775 |
|
S |
781 |
689 |
ADGPGLPAGSPQQPLSQPGSPAPGPPAQMDL |
9.94E-13 |
0.998507 |
|
|
S |
785 |
693 |
GLPAGSPQQPLSQPGSPAPGPPAQMDLEHPP |
3.03E-13 |
0.999999 |
pAb785 |
|
S |
867 |
775 |
SADFKEPPSLPGKEKSPPAAAAYGPPLTPQP |
0.000807143 |
1 |
pAb867 |
|
T |
879 |
787 |
KEKSPPAAAAYGPPLTPQPSPLSELPQAAPP |
0.0101532 |
1 |
pAb879 |
|
S |
883 |
791 |
PPAAAAYGPPLTPQPSPLSELPQAAPPPGSP |
1.36E-08 |
1 |
|
|
S |
897 |
807 |
PSPLSELPQAAPPPGSPTLPGRLEDFLESST |
1.92E-15 |
0.999573 |
pAb897/9 |
|
T |
899 |
809 |
PLSELPQAAPPPGSPTLPGRLEDFLESSTGL |
n/a |
n/a |
pAb897/9 |
|
S |
941 |
849 |
PLSLIDDLHSQMLSSSAILDHPPSPMDTSEL |
0.000584126 |
0.569645 |
|
|
S |
949 |
857 |
HSQMLSSSAILDHPPSPMDTSELHFAPEPSS |
8.45E-22 |
1 |