Chicken progesterone receptor expressed in Saccharomyces cerevisiae is correctly phosphorylated at all four Ser-Pro phosphorylation sites.
This study describes the phosphorylation of chicken progesterone receptor (cPR) produced in yeast, Saccharomyces cerevisiae, and examines the dependence of specific phosphorylations on hormone and DNA binding. The chicken progesterone receptor is expressed in vivo as two forms, cPRB and a smaller form, cPRA. Characterization of the phosphorylation sites in the cPRB form expressed in yeast shows that progesterone receptor is phosphorylated on the three serines (Ser211, Ser260, and Ser530) reported previously in chicken oviduct. An additional site which was phosphorylated in response to hormone was also detected and was subsequently identified as Ser367. Although cPRB and cPRA are phosphorylated identically in chicken oviduct, cPRA expressed in yeast is phosphorylated on Ser211, Ser260, and Ser367, but phosphorylation of Ser530 is almost undetectable. In contrast, cPRB expressed in yeast is phosphorylated on all four sites. No phosphorylations were found in or near the region required for hormone binding, indicating that phosphorylation is not required for hormone binding. In order to determine whether any of the phosphorylations were DNA-dependent, phosphorylation was also studied using cPRA containing a partial deletion of the DNA binding domain. Two of the sites, Ser211 and Ser367, showed reduced phosphorylation in this mutant, suggesting a possible requirement for DNA binding activity for the phosphorylation of these sites. To our knowledge, this is one of the first demonstrations that a eucaryotic protein expressed in yeast is correctly phosphorylated.
Poletti
A
A
Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.
Conneely
O M
OM
McDonnell
D P
DP
Schrader
W T
WT
O'Malley
B W
BW
Weigel
N L
NL
D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D003001:Cloning, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D011980:Receptors, Progesterone / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013329:Structure-Activity Relationship
D010750:Phosphoproteins; D011980:Receptors, Progesterone; D011994:Recombinant Proteins