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Title: "PHOSPHORYLATION OF SUCROSE SYNTHASE AT SERINE 170: OCCURRENCE AND POSSIBLE ROLE AS A SIGNAL FOR PROTEOLYSIS"

Author
item Hardin, Shane
item TANG, G - NC STATE UNIV
item SCHOLZ, A - NC STATE UNIV
item HOLTGRAEWE, D - NC STATE UNIV
item WINTER, H - NC STATE UNIV
item Huber, Steven

Submitted to: Plant Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/5/2003
Publication Date: 9/1/2003
Citation: Hardin, S.C., Tang, G.Q., Scholz, A., Holtgraewe, D., Winter, H., Huber, S.C. 2003. "Phosphorylation of Sucrose Synthase at Serine 170: Occurrence and Possible Role As a Signal for Proteolysis" [abstract]. Plant Journal. 35:588-603.

Interpretive Summary: The metabolism of sucrose in growing plant organs such as roots and developing seeds is of crucial importance to growth and development and ultimately, agricultural productivity. Our studies center on the enzyme sucrose synthase (SuSy) whose contribution to plant growth and development is very well established. Metabolism of imported sucrose through SuSy is correlated with "sink strength" and the partitioning of carbon into cellulose or starch. During investigations of SuSy regulation via phosphorylation we discovered a new, secondary site at serine-170. Contrary to our initial predictions, this modification was not directly involved in altered enzymatic activity, but instead appeared to "tag" the protein for degradation. This selective proteolytic turnover mechanism may regulates SuSy protein abundance and hence activity during development. In addition, the "26S proteasome" was identified as the likely proteolytic machinery involved in SuSy degradation, and an initial characterization of this complex in maize tissues is provided. Collectively, the results obtained provide the foundation for a new paradigm in regulation of an important enzyme of sucrose metabolism that may have far-reaching implications to include regulation of sink strength during leaf, nodule and fruit development.

Technical Abstract: Sequence analysis identified serine-170 (S170) of the maize (Zea mays L.) SUS1 sucrose synthase (SUS) protein as a possible, second phosphorylation site. Maize leaves contained two calcium-dependent protein kinase activities and a calcium-independent kinase activity with characteristics of an SNF1-related protein kinase. Phosphorylation of the novel S170 and the known S15 site by these protein kinases was determined in peptide substrates, and detected in SUS1 protein substrates utilizing sequence- and phosphorylation-specific antibodies. We demonstrate phosphorylation of S170 in vitro and in vivo. The calcium-dependent protein kinases phosphorylated both S170 and S15, whereas SNF1-related protein kinase activity was restricted to S15. Calcium-dependent protein kinase-mediated S170 and S15 phosphorylation kinetics were determined in wild type and mutant SUS1 substrates. These analyses revealed that kinase specificity for S170 was 3-fold lower than S15, and that phosphorylation of S170 was stimulated by prior phosphorylation at the S15 site. The SUS-binding peptides encoded by ENOD40 (Rohrig et al., 2002 PNAS, 99, 1915-1920) specifically antagonized S170 phosphorylation in vitro. A model wherein S170 phosphorylation functions as part of a mechanism targeting SUS for proteasome-mediated degradation is supported by the observations that SUS proteolytic fragments: 1) were detected and possessed relatively high pS170 stoichiometry; 2) were spatially coincident with proteasome activity within developing leaves; and 3) co-sedimented with proteasome activity. In addition, full-length pS170-SUS protein was less stable than S170-SUS in cultured leaf segments and was stabilized by proteasome inhibition. Post-translational control of SUS protein level through pS170-promoted proteolysis may explain the specific and significant decrease in SUS abundance that accompanies the sink-to-source transition in developing maize leaves.