L. Drzymala et al., Cellular phosphorylation of an acidic proline-rich protein, PRP1, a secreted salivary phosphoprotein, BIOCHEM, 39(8), 2000, pp. 2023-2031
Phosphorylation of many secreted salivary proteins is necessary for their b
iological functions. Identification of the kinase, which is responsible for
in vivo phosphorylation, is complicated, because several of the protein ph
osphorylation sites conform both to the recognition sequence of casein kina
se 2 (CK2) and Golgi kinase (G-CK), which both are found in the secretory p
athway. This study was undertaken to determine the kinase recognition seque
nce in a secreted proline-rich salivary protein, PRP1, and thereby identify
the responsible kinase. This was done by transfecting a human submandibula
r cell line, HSG, and a kidney cell line, HEK293, with expression vectors e
ncoding wild-type or mutated PRP1. It was shown that phosphorylation occurr
ed only at the same sites, Ser8 and 22, as in PRP1 purified from saliva. Ph
osphorylation at either site did not depend on the other site being phospho
rylated. 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 no
t conform to any known kinase recognition sites. If Ser22 was mutated to Th
r, no phosphorylation was seen, and a cluster of negatively charged residue
s 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 substrat
e sequence or by a novel kinase. No difference in phosphorylation was seen
between undifferentiated and differentiated HSG cells.