Poster: Protein modification

Abs # 808: Autophosphorylation Site Identification of CPK5, a Calcium-Dependent Protein Kinase

Presenter:	Harmon, Alice C., harmon@botany.ufl.edu
Authors	Strachan, Camille N. (A)   Hrabak, Estelle M. (D)   Stevens, Stanley  (B)   Winefordner, James D. (A)   Denslow, Nancy D. (B)   Harmon, Alice C. (C)
Affiliations:	(A): Dept. of Chemistry, University of Florida (B): Interdisciplinary Center for Biotechnology, University of Florida (C): Dept. of Botany, University of Florida (D): Dept of Plant Biology, University of New Hampshire

Calcium-dependent protein kinases (CDPKs) are found in vascular and nonvascular plants, green algae, and in certain protozoa. In Arabidopsis, CDPKS are encoded by 34 genes. Because the autophosphorylation properties of CDPKs are not fully understood, localization of the in vitro autophosphorylation sites of CPK5, a model member of this family, was determined by complimentary methods. Glutathione-S-transferase tagged CPK5 was autophosphorylated by incubation for one hour at room temperature in a Ca²⁺ buffer containing MgCl₂, EGTA, CaCl₂, molybdate and vanadate and the addition of ATP to 1 mM. The protein was digested with trypsin and analyzed by three mass spectrometers: an LCQ-Deca ion trap, a QSTAR, and MALDI-TOF. Different analysis techniques were employed for each of these instruments. Included in these methods is a novel, simple, cost effective method for phosphorylation identification. Results show the identification of at least five autophosphorylation sites by these three methods. The complimentary results of these methods gave an increased identification of autophosphorylation sites that would not have been possible by any one method. The combined use of these methods may be applied for the identification of phosphorylation sites for all phosphoproteins. Also, with the development of a simple, cost effective method for phosphorylation identification, many laboratories lacking the availability of expensive equipment will be able to apply this method. The increased coverage of autophosphorylation sites will result in a greater understanding of the importance of this mechanism in these kinases.