title: Domain analysis of a groundnut calcium-dependent protein kinase: nuclear localization sequence in the junction domain is coupled with nonconsensus calcium binding domains.
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.
authors.affiliation: Department of Biochemistry, Calcutta University, 35 Ballygunge Circular Road, Calcutta 700019.
authors.forename: Ayan
authors.initials: A
authors.lastname: Raichaudhuri
authors.forename: Rajasri
authors.initials: R
authors.lastname: Bhattacharyya
authors.forename: Shubho
authors.initials: S
authors.lastname: Chaudhuri
authors.forename: Pinak
authors.initials: P
authors.lastname: Chakrabarti
authors.forename: Maitrayee
authors.initials: M
authors.lastname: Dasgupta
mesh_terms: D000486:Allergy and Immunology; D000595:Amino Acid Sequence; D010367:Arachis / Q000378:metabolism; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D005982:Glutathione Transferase / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals; D009754:Nuts; D010455:Peptides / Q000737:chemistry; D010770:Phosphotransferases / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D011494:Protein Kinases / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D013347:Subcellular Fractions / Q000378:metabolism; D014466:Ultraviolet Rays; D017871:Calcium-Calmodulin-Dependent Protein Kinases
chemical_list: D002118:Calcium; D005982:Glutathione Transferase; D019913:Nuclear Localization Signals; D010455:Peptides; D010770:Phosphotransferases; D011494:Protein Kinases; D011994:Recombinant Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases