Evolutionary Adaptations of Plant AGC Kinases: From Light Signaling to Cell Polarity Regulation

Abstract

Signaling and trafficking over membranes involves a plethora of transmembrane proteins that control the flow of compounds or relay specific signaling events. Next to external cues, internal stimuli can modify the activity or abundance of these proteins at the plasma membrane (PM). One such regulatory mechanism is protein phosphorylation by membrane-associated kinases, several of which are AGC kinases. The AGC kinase family is one of seven kinase families that are conserved in all eukaryotic genomes. In plants evolutionary adaptations introduced specific structural changes within the AGC kinases that most likely allow modulation of kinase activity by external stimuli (e.g., light). Starting from the well-defined structural basis common to all AGC kinases we review the current knowledge on the structure-function relationship in plant AGC kinases. Nine of the 39 Arabidopsis AGC kinases have now been shown to be involved in the regulation of auxin transport. In particular, AGC kinase-mediated phosphorylation of the auxin transporters ABCB1 and ABCB19 has been shown to regulate their activity, while auxin transporters of the PIN family are located to different positions at the PM depending on their phosphorylation status, which is a result of counteracting AGC kinase and PP6 phosphatase activities. We therefore focus on regulation of AGC kinase activity in this context. Identified structural adaptations of the involved AGC kinases may provide new insight into AGC kinase functionality and demonstrate their position as central hubs in the cellular network controlling plant development and growth.

Keywords: AGC kinase; Arabidopsis thaliana; PIN polarity; evolution; kinase structure; phototropic growth.

Figure 1

Basic structure of AGCVIII kinases. (A) Two dimensional structure indicating the different protein domains of AGCVIII kinases. The N- and C-lobe (yellow or cyan) of the catalytic core are supplemented by regulatory domains in the N-tail (red), activation segment (magenta), and C-tail (green). These three domains are highly variable and their minimal and maximal lengths in AGCVIII kinases are denoted [N-tail 25–671 amino acids (AA), activation segment 55–137 AA, C-tail 33–87 AA]. Importantly, the magnesium binding loop (DFDLS) and the activation loop (SxSFVGTxEY) are separated by a 36–118 AA long insertion domain that is found in plant AGC kinases only. (B) Exemplary three dimensional structure of an AGC kinase with the same color coding as in the two dimensional structure. The depicted structure is based on a ternary crystal structure of mouse PKA [PDB-ID: 4DG2 (Bastidas et al., 2012)]. The beta-sheet rich N-lobe (yellow) is connected to the alpha-helix rich C-lobe (cyan) by a linker formed by subdomain V. Together they form the catalytic core of AGC kinases and enable ATP (black), magnesium (orange spheres), and substrate (not shown) binding in the catalytic cleft between the lobes. The activity of AGC kinases is regulated by post-translational modification of residues in, or binding of interactors to the N-tail (red), activation segment (magenta), or C-tail (green). The N-tail depicted here represents the anchor of a, in some cases much bigger, regulatory domain that extends from the core structure. Also the activation segment and the C-tail are highly diverse. The activation segment starts with a magnesium binding loop (DFDLS, black) and ends with the P + 1 loop (APE, purple) that is involved in substrate binding. Different from the depicted situation in PKA, a quite variable insertion domain of up to 118 AA can be found within the activation segment of plant AGC kinases. Likewise, the C-tail of most plant AGC kinases contains conserved elements, such as the PxxP motif or the PIF at its C-terminus (green), but also shows great diversity in composition and size.

Figure 2

Model for blue light (BL)-dependent regulation of phot1 activity. In the dark phot1 is unphosphorylated and associates to the PM. NPH3 on the other hand is phosphorylated by an unknown protein kinase (PK) and remains in the cytosol. BL triggers autophosphorylation of phot1, and this leads to dephosphorylation of NPH3 by an unidentified phot1-controlled protein phosphatase. Dephosphorylated NPH3 heterodimerizes with CUL3 and forms the CRL3^NPH3 E3 ubiquitin ligase. In this ligase NPH3 functions as anchor for the PHOT1 substrate. In low intensity BL (L-I BL) the activity of CRL3^NPH3 leads to mono-/multiubiquitination of PHOT1 and its subsequent localization to the cytoplasm. With higher BL intensities (H-I BL) CRL3^NPH3 mediates polyubiquitination of phot1. This marks phot1 for degradation by the 26S proteasome and eventually desensitizes the plant cell for BL by reducing the amount of available receptors. The phot1 kinase domain is colored yellow, and the photoreceptor domain is marked in blue. Phosphate groups on the proteins are indicated by red dots, while ubiquitin groups are represented by orange hexagons.

Figure 3

Regulation of ABCB auxin transporters by AGC kinases. (A) In darkness phot1 is inactive and does not phosphorylate ABCB19. In this state ABCB19 can interact with its positive modulator TWD1 and export auxin from the cell. (B) Blue light (BL) activates phot1 and induces phosphorylation of ABCB19. This inhibits ABCB19 – TWD1 interaction and blocks auxin efflux via ABCB19. (C) In the absence of TWD1, phosphorylation of a serine in the linker domain of ABCB1 by PID stimulates ABCB1 activity and leads to an increase of auxin export. (D) In the presence of TWD1, PID is no longer able to phosphorylate the linker domain of ABCB1. This lack of phosphorylation and potentially the phosphorylation of other sites of ABCB1 block ABCB1 activity and the respective auxin efflux. Red dots indicate phosphorylation events.

Figure 4

Model for phosphorylation-dependent re-localization of PINs. De novo synthesized PINs are distributed apolarly to the PM. Constitutive endocytosis relocates PINs depending on their phosphorylation status (red dots). This relocation might be baso-apical as in the figure (embryo, root, shoot, and inflorescence meristem), apolar-lateral (phototropism), between lobe and indentation (pavement cells), or between the inner or outer cell side (guard cells). Tuning of the activities of AGC kinases and PP6-type phosphatase determines the PIN phosphorylation status and thus the preferential targeting pathway, and leads to the respective redistribution of the PINs. Basal targeting of unphosphorylated PINs is GNOM-dependent; ARF-GEFs involved in other targeting pathways remain unknown. Grey arrows indicate trafficking of PIN-loaded vesicles, while black arrows indicate the direction of PIN-mediated auxin transport.