Serine/threonine/tyrosine phosphorylation of the LHX3 LIM-homeodomain transcription factor.
LHX3 is a LIM homeodomain transcription factor with essential roles in pituitary and motor neuron development in mammals. Patients with mutations in the LHX3 gene have combined pituitary hormone deficiency and other symptoms. In this study, we show that the LHX3 protein can be modified post-translationally by phosphorylation. LHX3 can serve as a substrate for protein kinase C and casein kinase II. Overexpression of these kinases reduces the transcriptional capacity of LHX3 to activate target genes. Following exposure of LHX3 to cellular kinases, mass spectrometry was used to map the phosphorylation of five amino acid residues within the human LHX3a isoform. Two phosphorylated residues (threonine 63 and serine 71) lie within the first LIM domain of the protein. Three other modified amino acids (tyrosine 227, serine 234, and serine 238) are located in the carboxyl terminus. Targeted replacement of these amino acids with non-modifiable residues significantly reduced the ability of LHX3 to activate both synthetic and pituitary hormone reporter genes. However, the amino acid replacements did not significantly affect the capability of LHX3 to interact with the NLI, PIT1, and MRG1 partner proteins, or its ability to bind to a high affinity DNA site. In conclusion, we have identified unique amino acids within LHX3 that are important for its transcriptional activity and are phosphorylated.
Parker
Gretchen E
GE
Department of Biology, Indiana University-Purdue University Indianapolis, Indianapolis, Indiana 46202-5132, USA.
West
Brooke E
BE
Witzmann
Frank A
FA
Rhodes
Simon J
SJ
D000595:Amino Acid Sequence; D002460:Cell Line; D015180:Electrophoresis, Gel, Two-Dimensional; D018398:Homeodomain Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D060850:LIM-Homeodomain Proteins; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000378:metabolism*; D013912:Threonine / Q000378:metabolism*; D014157:Transcription Factors; D014443:Tyrosine / Q000378:metabolism*
D018398:Homeodomain Proteins; D060850:LIM-Homeodomain Proteins; C094898:Lhx3 protein; D014157:Transcription Factors; D013912:Threonine; D014443:Tyrosine; D012694:Serine