AGCVIII kinases: at the crossroads of cellular signaling Yan Zhang and Sheila McCormick Plant Gene Expression Center, United States Department of Agriculture/Agricultural Research Service, and Department of Plant and Microbial Biology, University of California at Berkeley, 800 Buchanan St., Albany, CA 94710, USA AGCVIII kinases regulate diverse developmental and cellular processes in plants. As putative mediators of secondary messengers, AGCVIII kinases potentially integrate developmental and environmental cues into specific cellular responses through substrate phos- phorylation. Here we discuss the functionality and regu- lation of AGCVIII kinases. Specifically, we question the view that activities of AGCVIII kinases, like their animal counterparts, are regulated by a common regulator, 3-phosphoinositide-dependent protein kinase-1 (PDK1). Instead, increasing evidence suggests that Ca 2+ and phospholipids regulate AGCVIII kinases, by altering their activities or by affecting their subcellular localization. As AGCVIII kinases are at the crossroads of plant cellular signaling, they and the signaling networks in which they participate are keys to a better understanding of plant development and of interactions with their environ- ment. AGC kinases: mediators of cellular signaling Phosphorylation is the most common and universal way of regulating protein function in eukaryotes. Reversible phosphorylation regulates protein activity, subcellular localization, stability and interaction with other proteins [1,2]. The AGC kinases belong to one of the six serine/ threonine kinase superfamilies in plants [1]. AGC kinases are collectively named to include c AMP-dependent protein kinases (PKA), c GMP-dependent protein kinases (PKG), various types of protein kinase C (PKC), protein kinase B (PKB), 3-phosphoinositide-dependent protein kinase-1 (PDK1), and the ribosomal protein S6 kinases [3]. In animals and yeast, AGC kinases are critical mediators that perceive and interpret signaling initiated from sec- ondary messengers such as cAMP, cGMP, Ca 2+ and phos- pholipids through substrate phosphorylation [3]. Although most members of the plant AGC kinases have no assigned function, analysis of some AGC kinase mutants showed that they play essential roles in various cellular and devel- opmental processes (Table 1), such as root hair and pollen tube growth [4–7], auxin transport [8–11], light sensing [12,13] and abiotic and biotic stresses [14–16]. That plant AGC kinases are involved in diverse cellular and develop- mental processes, together with analogy to the roles played by their animal and yeast counterparts, indicates that plant AGC kinases are at key positions to initiate cellular responses upon perception of developmental cues and extracellular stimuli. Placing AGC kinases in the context of signaling networks by identifying their regulatory mech- anisms and downstream components will undoubtedly facilitate our general understanding of plant development and of interactions with their environment. Plant AGC kinases were initially characterized into several subfamilies, including AGCVIII kinases, AGCVII kinases, AGCVI kinases, AGC other, and homologs of animal PDK1 [17]. The largest AGC kinase subfamily, AGCVIII kinases, was later sub-divided into four groups, named AGC1-AGC4 [18]. In this article, we focus on the AGCVIII subfamily because our understanding of other AGC kinases has not been significantly advanced since their initial characterization in 2003 [17]. We discuss the molecular mechanisms underlying the functionality of AGCVIII kinases in light of recent findings. Specifically, Review Glossary Ade motif: A highly conserved motif of 4 to 5 amino acids, [FD(X) 1–2 Y/F], within the C-terminal tail of most animal AGC kinases, which is responsible for PDK1 binding. However, plant AGC kinases do not have this motif. EF-hand motif: A helix-loop-helix domain engaged in Ca 2+ -binding. An EF- hand motif is found in many Ca 2+ -regulated proteins such as calmodulin, Ca 2+ - dependent protein kinases and calcineurin B. C2 domain: A Ca 2+ and phospholipid-binding domain found in many types of proteins. Ca 2+ and phospholipid binding to C2 domains regulate protein subcellular targeting and activity. IP 3 : Inositol 1,4,5-trisphosphate, a soluble phospholipid regulating the release of Ca 2+ from internal stores such as the endoplasmic reticulum. It can be generated from another phospholipid PI(4,5)P 2 through the activity of phospholipase C, and can be dephosphorylated through the activity of inositol polyphosphate 5-phosphatases. LOV domain: Light, oxygen, voltage domain. Two LOV domains are located upstream of the catalytic domain of phototropins. Blue light induces phosphorylation changes of LOV domains and of sequences between the two LOV domains, leading to autophosphorylation and activation of photo- tropins. PA: Phosphatidic acid, a phospholipid generated either by phospholipase D or by diacylglycerol kinase. PI3P: Phosphatidylinositol 3-phosphate, a membrane phospholipid enriched at endosomes. PI3P regulates protein sorting at endosomes and regulates vesicle fusions at target membrane compartments by recruiting protein complexes. PIP 3 : Phosphatidylinositol 3,4,5-trisphosphate, a membrane phospholipid produced upon extracellular stimuli by the activity of class I PI3P kinases. PIP 3 is an instructive signal for actin polymerization. Plants do not have class I PI3P kinase. PIP 3 has not been detected in plants. PDK1: 3-phosphoinositide-dependent protein kinase-1. PDK1 is an evolutiona- rily conserved ser/thr kinase whose activity and subcellular localization can be regulated by specific phospholipids. Animal PDK1s bind to phosphatidylino- sitol 3,4,5-trisphosphate (PIP 3 ), which is absent in plants. Although Arabidopsis PDK1 showed binding to diverse phospholipids, only PA significantly increased PDK1 activity in vitro and in vivo. PIF: PDK1-interacting fragment. The PIF motif is a highly conserved hydro- phobic motif at the C-terminal tail of most AGC kinases, including plant AGC kinases. TLI: T-loop insertion sequence, a 50 to 80 amino acid sequence present in the activation loop of all plant AGCVIII kinases but absent in animal AGC kinases. A domain-swapping experiment showed that the TLI was critical for subcellular localization of AGCVIII kinases, likely through protein-protein interactions. Corresponding author: McCormick, S. (sheilamc@berkeley.edu). 1360-1385/$ – see front matter . Published by Elsevier Ltd. doi:10.1016/j.tplants.2009.09.006 Available online 7 October 2009 689