Pseudosubstrate Inhibition of CDPK, a Protein Kinase with a Calmodulin-like Domain

Abstract

Between the catalytic and regulatory domains of calmodulin-like domain protein kinase, CDPK, is a junction domain which has some identity to the autoinhibitory domain of calmodulin-dependent protein kinase type II (Harper, J. F., Sussman, M. R., Schaller, G. E., Putnam-Evans, C., Charbonneau, H., & Harmon, A. C. (1991) Science 252, 951-954). To investigate whether CDPK's junction domain also functions as an autoinhibitory domain, we determined the effect of synthetic peptides, corresponding to sequences within the junction domain, on the activity of native soybean CDPK. Three peptides, corresponding to residues 310-332, 318-332, 302-317, were competitive inhibitors with respect to syntide-2 and had Ki values of 5, 25, and 85 microM, respectively. These peptides were uncompetitive inhibitors with respect to ATP and had Ki values of 24, 220, and 510 microM, respectively. A fourth peptide, CDPK alpha 302-332, inhibited activity by a mixed mechanism with respect to both syntide-2 (Ki = 1.9 microM; K'i = 5.0 microM) and ATP (Ki = 15 microM; K'i = 4.5 microM). Three of the peptides, CDPK alpha 302-332, 310-332, and 318-332, formed complexes with soybean calmodulin during electrophoresis in native polyacrylamide gels and were able to inhibit calmodulin-dependent protein kinases. Given the similarity between CDPK's calmodulin-like domain and calmodulin (40% sequence identity), it was possible that these peptides could inhibit activity through interaction with the calmodulin-like domain rather than the catalytic domain. To address this possibility, a cDNA encoding the first 312 residues of soybean CDPK alpha was constructed and expressed in Escherichia coli. This enzyme, which is missing most of the junction domain and all of the calmodulin-like domain, was active in the presence and absence of calcium. Peptide CDPK alpha 310-332 inhibited this truncated enzyme competitively with respect to syntide-2 (Ki = 4 microM). These results show that the junction domain is capable of functioning as an autoinhibitory domain, possibly through a pseudosubstrate site located between residues 310 and 332.

Characterisation and cloning of a calmodulin-like domain protein kinase from Chlamydomonas moewusii (Gerloff )

“…7A, identical amino acids in the junction domain are boxed. The amino acids in this domain that have been implicated from studies with inhibitor peptides (Harmon et al 1994) and deletion mutants ) to be involved in regulating enzyme activity are conserved in the CCK1 junction domain, suggesting that its function is also conserved. When the kinase domains of CDPKs from higher plants and Chlamydomonas are compared with those of protein serine/threonine kinases, they clearly form a separate family (Fig.…”

A Single-Pore Residue Renders the Arabidopsis Root Anion Channel SLAH2 Highly Nitrate Selective

“…We therefore disrupted CPK21 and CIPK23 kinase activity by exchanging Asp-204 with Ala (CPK21DEFD204A) and Lys-60 with Asn (Harmon et al, 1994;Harper et al, 1994;Franz et al, 2011) and coexpressed SLAH2 alone or with either CPK21DEF, CPK21DEFD204A, CIPK23/CBL1, or CIPK23 K60N/CBL1. In this experiment, SLAH2-mediated anion currents were detected only in oocytes coexpressing SLAH2 with CPK21DEF or CIPK23/CBL1, but not in the absence of a kinase or with the kinase-inactive mutants ( Figure 1E).…”

Calcium‐dependent protein kinase from maize seedlings activated by phospholipids

“…Surprisingly, a peptide corresponding to the autoinhibitory domain (residues 302±332) of CDPKa [34,35] inhibited PKC with greater potency than it inhibited ZmCPKp54 (Fig. 6).…”