Abstract
The signature of calcium-dependent protein kinases (CDPKs) is a C-terminal calmodulin-like domain (CaMLD) with four consensus calcium-binding sites. A junction domain (JD) joins the kinase with CaMLD and interacts with them through its autoinhibitory and CaMLD binding subdomains, respectively. We noted several CDPKs additionally have a bipartite nuclear localization signal (NLS) sequence as a subdomain in their JD, and this feature is obligatorily coupled with the absence of consensus calcium-binding sites in their respective CaMLDs. These predicted features are substantiated by undertaking investigations on a CDPK (gi:67479988) isolated from cultured groundnut (Arachis hypogea) cells. This kinase can bind 3.1 mol of Ca(2+) under saturating conditions with a considerably high K(d) of 392 mum as compared with its canonical counterparts. CD spectroscopic analysis, however, indicates the intramolecular structural changes accompanied with calcium binding to be similar to canonical CDPKs. Attesting to the presence of NLS in the JD, the endogenous kinase is localized in the nucleus of osmotically stressed Arachis cells, and in vitro binding assays indicate the NLS in the JD to interact with nuclear transport factors of the importin family. Homology modeling also indicates the feasibility of interaction of importins with the NLS present in the JD of such CDPKs in their activated form. The possible significance of obligatory coupling between the presence of NLS in the junction domain and atypical calcium binding properties of these CDPKs is discussed in the light of the known mechanisms of activation of these kinases.
Highlights
CDPKs5 are unique transducers of calcium signaling in plants and protists that are absent in yeasts and animals [1,2,3]
Domain Analysis of an A. hypogea calcium-dependent protein kinases (CDPKs) and Its Homologs; Nuclear Localization Sequence in the junction domain (JD) Is Coupled with Nonconsensus EF Hand Loops in the calmodulin-like regulatory domain (CaMLD)—We developed a 1.3-kb partial cDNA clone of an Arachis CDPK by amplification of RNA prepared from Arachis cells that were subjected to osmotic stress [27, 28]
In addition to having the core domain arrangement of a canonical CDPK, AhCPK2 contains a bipartite nuclear localization signal (NLS) sequence (PROSITE code no PS00015) in its junction domain, and the sequence of the second calcium binding EF hand loop (PROSITE code PS00018) in its CaMLD was a deviation from the consensus (Fig. 2, A and B)
Summary
CDPKs5 are unique transducers of calcium signaling in plants and protists that are absent in yeasts and animals [1,2,3]. In addition to interaction of the CaMLD with its target in the junction domain, intramolecular activation of CDPKs involves a conformational change of the holoenzyme mediated through the tether. Genomic sequencing as well as several extensively expressed sequence tag projects indicate the presence of multigene families of CDPKs in various plants [2, 11] They are demonstrated to be involved in regulating ion transport and/or gene expression linked with various cellular processes like cytoskeleton dynamics, stress response, growth and development, and metabolism [3]. Such response specificity of individual CDPKs is believed to be generated by their varying subcellular compartmentalizations, varying calcium and lipid sensitivities, and differences in substrate recognition [2]. In the light of the known mechanism of regulation of CDPKs [5,6,7,8,9], we discuss the possible reasons for the essentiality of coupling between the presence of an NLS in the JD and the nonconsensus calcium-binding motifs in the CaMLD
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