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Title: IDENTIFICATION OF A NEW MOTIF FOR CDPK PHOSPHORYLATION IN VITRO THAT SUGGESTS ACC SYNTHASE MAY BE A CDPK SUBSTRATE

Author
item SEBASTIA, C - UNIV OF ILLINOIS-UC
item Hardin, Shane
item CLOUSE, S - NORTH CAROLINA STATE
item KIEBER, J - UNIV OF NORTH CAROLINA
item Huber, Steven

Submitted to: Archives of Biochemistry and Biophysics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/27/2004
Publication Date: 8/1/2004
Citation: Sebastia, C.H., Hardin, S.C., Clouse, S.C., Kieber, J.J., Huber, S.C. 2004. Identification of A New Motif for CDPK Phosphorylation In Vitro That Suggests ACC Synthase May Be A CDPK Substrate. Archives Of Biochemistry and Biophysics. 428:81-91.

Interpretive Summary: The calcium-dependent protein kinase (CDPK) superfamily is a large and important family of protein kinases that are unique to plants and are thought to control numerous aspects of growth and development. We demonstrate in the present study that CDPKs will recognize the variant motif: hydrophobic-Arg-hydrophobic-Ser/Thr-hydrophobic-X-Lys-Arg, where X is nominally any amino acid. Recognition of this new motif may explain phosphorylation of ACC synthase (ACS) at the known regulatory C-terminal phosphorylation site. These results suggest that CDPKs, and hence calcium signaling, may be involved in the control of ethylene biosynthesis. The results also have application to a broad range of other cellular proteins, as database searches suggest that many additional proteins contain sequences that conform to the new motif (referred to as the 'ACS motif') and thus may be phosphoproteins. There are three other important implications of this work. First, it is noteworthy that the closely related SnRK1 protein kinases, which also recognize the classic motif targeted by CDPKs, do NOT recognize the ACS motif. Thus, sequences that conform to the new motif may be selectively phosphorylated in response to calcium signaling (and not to metabolic signals that are thought to control SnRK1 activity). Secondly, the present results suggest that CDPKs may have a series of overlapping but non-identical polypeptide binding grooves that can accommodate the different sequences. Finally, we demonstrate that CDPKs (as well as SnRK1s) will not recognize a phosphorylation motif presented in reverse order. This is relevant to the present study because in some cases the ACS motif contains the classic motif in reverse order; if all that was involved in substrate recognition was interaction with side chains, then this could provide a simple explanation for the results. However, this is shown not to be the case (for the first time as far we are aware), which suggests that stereochemistry is critical. These results provide new information about the molecular basis for specificity of protein kinases, and thus will be valuable to a wide range of plant scientists. Understanding how protein kinases target their substrate proteins may provide rationale strategies for controlling the phosphorylation of plant proteins through site-directed mutagenesis. In addition, the results increase our ability to predict which proteins in crop plants are potentially controlled by phosphorylation, which is an important tool in functional genomics.

Technical Abstract: 1-Amino-cyclopropane-1-carboxylate synthase (ACS) catalyzes the rate-determining step in the biosynthesis of the plant hormone ethylene. Expression is controlled at the transcriptional level, and there is evidence for regulation of stability of the protein by reversible protein phosphorylation. The site of phosphorylation of the tomato enzyme, LeACS2, was recently reported to be Ser460, but the requisite protein kinase has not been identified. In the present study, a synthetic peptide based on the known regulatory phosphorylation site (KKNNLRLS460FSKRMY) in LeACS2 was found to be readily phosphorylated in vitro by several calcium-dependent protein kinases (CDPKs), but not a plant SNF1-related protein kinase or the kinase domain of the receptor-like kinase, BRI1, involved in brassinosteroid signaling. Phosphorylation of the LeACS2-Ser460 peptide by CDPKs was surprising because the sequence lacks a basic residue at P-3/P-4 (relative to the phosphorylated Ser at position P) and a hydrophobic group at P-5, both of which are considered essential recognition elements in the classic motif targeted by CDPKs. We demonstrate that phosphorylation of the LeACS2-Ser460 peptide is most dependent on basic residues at P+3/P+4 and hydrophobic residues at P-1 and P+1. The results establish a fundamentally new phosphorylation motif that is broadly targeted by CDPKs: hydrophobic-[ST]-hydrophobic-X-Basic-Basic. The results also suggest that CDPKs are involved in the phosphorylation of ACS, and thus, control of ethylene biosynthesis may involve calcium signaling. Database analysis using the new motif predicts a number of novel phosphorylation sites in plant proteins. Finally, we also demonstrate that CDPKs and SnRK1s do not recognize motifs presented in the reverse order, indicating that side chain interactions alone are not sufficient for substrate recognition.