Cellular phosphorylation of an acidic proline-rich protein, PRP1, a secreted salivary phosphoprotein.
Phosphorylation of many secreted salivary proteins is necessary for their biological functions. Identification of the kinase, which is responsible for in vivo phosphorylation, is complicated, because several of the protein phosphorylation sites conform both to the recognition sequence of casein kinase 2 (CK2) and Golgi kinase (G-CK), which both are found in the secretory pathway. This study was undertaken to determine the kinase recognition sequence in a secreted proline-rich salivary protein, PRP1, and thereby identify the responsible kinase. This was done by transfecting a human submandibular cell line, HSG, and a kidney cell line, HEK293, with expression vectors encoding wild-type or mutated PRP1. It was shown that phosphorylation occurred only at the same sites, Ser8 and 22, as in PRP1 purified from saliva. Phosphorylation at either site did not depend on the other site being phosphorylated. The sequence surrounding Ser8 has characteristics of both CK2 and G-CK recognition sequences, but destruction of the CK2 recognition site had no effect on phosphorylation, whereas no phosphorylation occurred if the G-CK recognition sequence was altered. The sequence surrounding Ser22 did not conform to any known kinase recognition sites. If Ser22 was mutated to Thr, no phosphorylation was seen, and a cluster of negatively charged residues at positions 27-29 was identified as part of the enzyme recognition site. Ser22 may be phosphorylated by a G-CK that recognizes an atypical substrate sequence or by a novel kinase. No difference in phosphorylation was seen between undifferentiated and differentiated HSG cells.
Drzymala
L
L
Department of Biochemistry, University of Toronto, Toronto M5S 1A8, Canada.
Castle
A
A
Cheung
J C
JC
Bennick
A
A
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D047390:Casein Kinase II; D002460:Cell Line; D003094:Collagen / Q000737:chemistry; D004338:Drug Combinations; D006801:Humans; D007668:Kidney / Q000737:chemistry; D007797:Laminin / Q000737:chemistry; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010455:Peptides / Q000737:chemistry*; D010710:Phosphates / Q000737:chemistry; D010766:Phosphorylation; D011233:Precipitin Tests; D055232:Proline-Rich Protein Domains; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry; D011509:Proteoglycans / Q000737:chemistry; D012469:Salivary Glands / Q000737:chemistry* / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000378:metabolism; D014162:Transfection
D004338:Drug Combinations; D007797:Laminin; D010455:Peptides; D010710:Phosphates; D011509:Proteoglycans; C059485:matrigel; D012694:Serine; D003094:Collagen; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases