Fig. 9

Identification of the PS-2 phosphorylation sites and potential candidate kinases

(A) Sequence comparison of the N-terminal domain of PS-1 and PS-2. Three serine residues (S) are located within a stretch of acidic amino acids (underlined by a striped bar) of PS-2 which is absent in PS-1. The three serine residues are potential substrates for CK-2 (Ser 7 and Ser 9) and CK-1 (Ser 19). (B) COS-7 cells transfected with the cDNA constructs PS-2Δas (which deletes the entire acid stretch shown in Panel A) and PS-2 Ser7/9Ala (which exchanges serine residues 7 and 9 by alanine) were metabolically labeled with [³⁵S]-methionine or [³²P]-orthophosphate and immunoprecipitated with antibody BOS4627. The deletion of the acidic stretch significantly inhibits phosphate incorporation, whereas single point mutations at serine residues 7 and 9 result only in a partial reduction of phosphate incorporation. (C) Quantitation of the results shown in Panel B. The deletion of the acidic stretch results in an almost complete inhibition of phosphate incorporation, whereas the two point mutations at serine residues 7 and 9 remove only two-thirds of the phosphate incorporation. Bars = mean ± SEM of three to four independent experiments. (D) GST fusion proteins with (NT PS-2 GST) or without the N terminus of PS-2 (GST) were phosphorylated in vitro with recombinant CK-1 and CK-2. Both enzymes phosphorylated the GST fusion protein with the N terminus of PS-2, whereas no phosphorylation was observed if GST alone was added to the in vitro assay. Arrowhead indicates autophosphorylation of the β-subunit of CK-2 (43).