Dissertation/Thesis Abstract

The GRIK-SnRK1 Kinase Cascade: Characterizing Phosphorylation of TCP Transcription Factors and Motifs in Arabidopsis Proteins
by Greeley, Laura Ann, Ph.D., North Carolina State University, 2016, 199; 10585498
Abstract (Summary)

Abiotic and biotic stresses often reduce the yields of native plants and economically important crop plants significantly. Due to their sessile nature, plants have evolved complicated response pathways to react to environmental stresses and signals. SNF1-related kinase 1 (SnRK1) is the plant homolog of mammalian AMPK (AMP-activated protein kinase) and yeast SNF1 (Sucrose Non-fermenting-1). SnRK1 is part of a protein kinase cascade that includes the GRIKs (Geminivirrus Rep Interacting Kinases), which activate SnRK1 by phosphorylating its activation loop. SnRK1 is critical for the regulation of many plant signaling pathways including global regulation of metabolism and energy conservation, and responses to abiotic and biotic stresses. SnRK1 also plays a role in regulation of plant development, most likely by modulating transcription factors to alter transcriptional gene expression programs.

Teosinte branched 1/Cycloidea/PROLIFERATING CELL FACTORS (TCPs) are a family of transcription factors that are unique to green algae and plants. TCPs fall into two classes that play opposing roles in plant development. Class I TCPs promote cell proliferation early in plant development, while Class II TCPs inhibit proliferation and promote differentiation. Although the knowledge about the functions of TCPs and the regulation of their mRNAs have been accumulating, very little is known about regulation of the proteins themselves.

The work described in this thesis characterized TCP phosphorylation by the GRIKSnRK1 cascade. These studies established that the majority of TCPs are phosphorylated by GRIK and/or by GRIK-activated SnRK1 in vitro. Analysis of truncated proteins revealed that TCP3 is phosphorylated in its C-terminus after the TCP domain. A combination of mass spectroscopy and site-directed mutagenesis showed that SnRK1 phosphorylates TCP18 on Ser-36 or Ser-38 and Thr-233, TCP20 on Ser-237, and TCP22 on Ser-140. All of these sites are also located outside of the TCP domain. Comparison of wild-type TCP20 and TCP22 with their corresponding phosphomutants and phosphomimics at Ser-237 and Ser-140, respectively, revealed that SnRK1 phosphorylation does not impact protein-DNA binding in vitro. In contrast, TCP20 and TCP22 phosphomimics were impaired for binding to BRM, a subunit of the SWI/SNF chromatin remodeling complex. These results suggested that SnRK1 phosphorylation of TCP20 and TCP22 regulates their transcription activity by altering their protein interactions.

This thesis also describes a proteomic approach to reveal a more specific consensus phosphorylation motif for SnRK1. This strategy used LC/MS/MS analysis to identify phosphopeptides derived from Arabidopsis culture cell lysates incubated with GRIKactivated SnRK1 in vitro. The sequence -xxxRSxsFxxxxx- was identified as a motif for GRIK-SnRK1 cascade phosphorylation. This motif is a more specific variation of one of the motifs in the literature.

Indexing (document details)
Advisor: Hanley-Bowdoin, Linda
School: North Carolina State University
School Location: United States -- North Carolina
Source: DAI-B 78/08(E), Dissertation Abstracts International
Subjects: Plant sciences, Biochemistry, Developmental biology
Keywords: Phosphorylation, Plant development, Plant transcription factors
Publication Number: 10585498
ISBN: 9781369641523