TABLE 2.
Phosphorylation sites flanking the basic region of the POU homeodomain
| Protein | Region
|
||
|---|---|---|---|
| Linker | Basic | Helix 1 | |
| Pit-1 | QVGALYNEKVGANE | RKRKRRTTI | SIAAKDALERHFG |
| Oct1 | SPSALNSPGIEGLS | RRRKKRTSI | ETNIRVALEKSFL |
| Oct2 | NQLSSPSLGFEPAG | RRRKKRTSI | ETNVRFALEKSFL |
| cfla1 | TGSPTSIDKIAAQG | RKRKKRTSI | EVSVKGALEQHFH |
| Brn-1 | TGSPTSIDKIAAQG | RKRKKRTSI | EVSVKGALESHFL |
| unc-86 | KDTIGDINGILPNT | DKKRKRTSI | AAPEKRELEQFFK |
| Brn-3 | QREKMNKPELFNGG | EKKRKRTSI | AAPEKRSLEAYFA |
| i-pou | KRRDPDAPSVLPAG | EKK--RTSI | AAPEKRSLEAYFA |
| Oct3/4 | NLQEICKSETLVQA | -RKRKRTSI | ENRVRWSLETMFL |
| TCFβ1 | GQQNLMEFVGGEPS | KKRKRRTSF | TPQAIEALNAYFE |
| § | †† | § | |
The POU domain protein sequences in the linker-basic-helix 1 region of the DNA binding homeodomain are shown. The flanking threonine and serine residues in the basic region which have been shown by others (4, 21, 38) to be phosphorylated in Oct1 and Pit1 are identified by daggers. The two sites in TCFβ1, one in the linker region and the other in helix 1, which are phosphorylated by JNK2 are identified by “§”. This was shown by making synthetic peptides corresponding to this region of TCFβ1 and determining whether they were phosphorylated by JNK2 in vitro. We generated three TCFβ1 peptides. The wild-type peptide (VGGEPS KKRKRRTSF TPQAI) was phosphorylated by JNK2. Mutant peptide A (VGGEPS KKRKRRAAF TPQAI), in which residues in the basic region that are phosphorylated in Oct1 and Pit1 are mutated, was found to be still phosphorylated by JNK2 in vitro. Mutant peptide B (VGGEPA KKRKRRTSF APQAI), in which the two potential JNK2 sites are mutated, was not phosphorylated by JNK2 in vitro. The mutated residues (underlined) involve substitution of alanine for serine or threonine.