Antagonistic Regulation of PIN Phosphorylation by PP2A and PINOID Directs Auxin Flux

Abstract

In plants, cell polarity and tissue patterning are connected by intercellular flow of the phytohormone auxin, whose directional signaling depends on polar subcellular localization of PIN auxin transport proteins. The mechanism of polar targeting of PINs or other cargos in plants is largely unidentified, with the PINOID kinase being the only known molecular component. Here, we identify PP2A phosphatase as an important regulator of PIN apical-basal targeting and auxin distribution. Genetic analysis, localization, and phosphorylation studies demonstrate that PP2A and PINOID both partially colocalize with PINs and act antagonistically on the phosphorylation state of their central hydrophilic loop, hence mediating PIN apical-basal polar targeting. Thus, in plants, polar sorting by the reversible phosphorylation of cargos allows for their conditional delivery to specific intracellular destinations. In the case of PIN proteins, this mechanism enables switches in the direction of intercellular auxin fluxes, which mediate differential growth, tissue patterning, and organogenesis.

Inactivation of Plasma Membrane–Localized CDPK-RELATED KINASE5 Decelerates PIN2 Exocytosis and Root Gravitropic Response inArabidopsis

“…In fact, we observed that the crk5-1 mutation not only reduces the amount of PM-associated PIN2 in the epidermis of transition zone, but also markedly accelerates brefeldin-sensitive internalization of PIN2 in epidermal cells. Subsequently, we found that CRK5 phosphorylates in vitro the central hydrophilic T-loop of PIN2, which modulates its PM association and stability in the epidermis (Michniewicz et al, 2007;Dhonukshe et al, 2010). In conclusion, these results suggest that CRK5 phosphorylation is specifically involved in the regulation of brefeldin-sensitive membrane recycling of PIN2 in the root transition zone.…”

“…The altered polar localization of PIN2 in cells of the transition zone and the generally reduced amount of PIN2 in epidermal and cortex cell membranes of crk5-1 roots compared with the wild type suggested a potential defect in membrane recycling leading to destabilization of PIN2 (Michniewicz et al, 2007;Dhonukshe et al, 2010). Similarly to the effects of phosphatidylinositol monophosphate 5-kinase 2 (Mei et al, 2012), we found that BFA treatment stimulated faster internalization of PIN2-GFP from epidermal cell membranes of crk5-1 roots compared with the wild type ( Figures 8A to 8H).…”

“…Without changing apical localization of PIN2 in the epidermis, low-level BFA treatment stimulates basal to apical PIN2 transcytosis in the cortex, which abolishes root gravitropic response, demonstrating the importance of cortical auxin recycling to the root tip (Rahman et al, 2010). Basal-toapical switching of PINs is stimulated by the PINOID (PID), WAG1, and WAG2 AGC kinases, which phosphorylate TPRxS motifs in the hydrophilic loops of PINs (Michniewicz et al, 2007;Dhonukshe et al, 2010). Phosphorylated PINs are recycled from the apical membrane by clathrin-dependent endocytosis controlled by the ARA7 GTPase and brefeldin-insensitive GNOM-LIKE1 proteins and their regulatory partners (reviewed in Löfke et al, 2013).…”

Embryogenesis: Pattern Formation from a Single Cell

“…This interpretation, originally derived from auxin application experiments is well supported by auxin transport visualization studies in embryos (Benkova et al, 2003;Michniewicz et al, 2007). Prior to cotyledon outgrowth, the PIN1 protein is preferentially localized to lateral membranes of cells in the epidermal (L1) layer in a pattern consistent with the transport of auxin toward the incipient primordia ( Figure 7 and Figure 14E; Benkova et al, 2003;Friml et al, 2003;Heisler et al, 2005).…”

“…Further evidence for a critical role for auxin localization by PIN1 is provided by analysis of the PID1 gene, which appears to be required for correct redistribution of PIN1 in the apex at the transition from globular to heart stage. Evidence suggests that the PID1 AGC kinase mediates the membrane localization of PIN proteins by phosphorylating the central hydrophilic loop (Michniewicz et al, 2007). This process is antagonized by PP2A phosphatase, which is required for normal embryonic and seedling development (Michniewicz et al, 2007).…”