Phosphorylation of a plant protease inhibitor protein by wheat calcium-dependent protein kinase

Abstract

Wheat germ ( Triticum aestivum) Ca 2+-dependent protein kinase (CDPK) phosphorylates soybean ( Glycine max) Bowman-Birk trysin/chymotrypsin inhibitor protein (BBI) and [ 32P]phospho BBI comigrates with BBI on SDS-PAGE. The phosphorylation of S 34 within the sequence RLNSCHS 34ACK was determined from isolation of [ 32P]-phospho-LNSCHSACK from tryptic digests of [ 32P]-phosphoBBI and from subsequent analysis of the Edman degradation products. Confirmation of S 34 phosphorylation was obtained from similar analysis of phosphopeptides purified from the tryptic digest of 2-bromoethylamine-derivatised [ 32P]phosphoBBI. This phosphorylation site differs from the Basic-X-X-Ser(Thr) phosphorylation site specificity previously found for synthetic peptide substrates of wheat CDPK. The adjacent S 31 of BBI, which does lie within a Basic-X-X-Ser motif, is not phosphorylated. Bovine serum albumin (BSA) is also phosphorylated by wheat germ CDPK on a site not involving a Basic-X-X-Ser motif, namely on S 468 within the sequence K 464TPVSEK 470. The site of phosphorylation of BBI by CDPK is located in an exposed central loop lying between the two anti-parallel β-sheet protease inhibitory domains. Two other CDPK protein substrates have phosphorylation site sequences of either Ser-X-X-Basic or Ser-X-Basic as found for BBI and BSA, respectively, suggesting that a C-terminal side basic residue could be a substrate recognition determinant for plant CDPK on some protein substrates.