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

Abstract

CRK5 is a member of the Arabidopsis thaliana Ca 2+ /calmodulin-dependent kinase-related kinase family. Here, we show that inactivation of CRK5 inhibits primary root elongation and delays gravitropic bending of shoots and roots. Reduced activity of the auxin-induced DR5-green fluorescent protein reporter suggests that auxin is depleted from crk5 root tips. However, no tip collapse is observed and the transcription of genes for auxin biosynthesis, AUXIN TRANSPORTER/AUXIN TRANSPORTER-LIKE PROTEIN (AUX/LAX) auxin influx, and PIN-FORMED (PIN) efflux carriers is unaffected by the crk5 mutation. Whereas AUX1, PIN1, PIN3, PIN4, and PIN7 display normal localization, PIN2 is depleted from apical membranes of epidermal cells and shows basal to apical relocalization in the cortex of the crk5 root transition zone. This, together with an increase in the number of crk5 lateral root primordia, suggests facilitated auxin efflux through the cortex toward the elongation zone. CRK5 is a plasma membrane-associated kinase that forms U-shaped patterns facing outer lateral walls of epidermis and cortex cells. Brefeldin inhibition of exocytosis stimulates CRK5 internalization into brefeldin bodies. CRK5 phosphorylates the hydrophilic loop of PIN2 in vitro, and PIN2 shows accelerated accumulation in brefeldin bodies in the crk5 mutant. Delayed gravitropic response of the crk5 mutant thus likely reflects defective phosphorylation of PIN2 and deceleration of its brefeldin-sensitive membrane recycling.

Members of the Plant CRK Superfamily Are Capable of Trans- and Autophosphorylation of Tyrosine Residues

“…However, cytoplasmic localization might also be possible because we did not compare its localization with a plasma membrane-localized marker protein. Among the CRK family members, six CRKs (1, 2, 3, 5, 7, and 8) were predicted to have a myristoylation/palmitoylation motif in the N terminus (29) and a predicted nuclear localization signal motif was found in all CRKs (59). In fact, tomato CRK1 and CPK/CRK-related tobacco CPK5 that have a myristoylation/palmitoylation motif in the N-terminal are localized to the plasma membrane, whereas motif mutants of these are localized to the nucleus and cytoplasm (60,61).…”

The dynamics of plant plasma membrane proteins: PINs and beyond

“…PID activation is predicted to promote apicalization of PINs, analogous to phenotypes observed in supo1 and in Ca 2+ -ATPase silencer lines (Friml et al, 2004;Zhang et al, 2011). In addition, an Arabidopsis Ca 2+ /calmodulin-dependent kinase-related kinase 5 (CRK5) lossof-function mutant demonstrated deficiencies in apical protein sorting, specifically of PIN2 in the root epidermis and in cortical root meristem cells (Rigo et al, 2013). This underscores the role of calcium in PIN polar targeting, suggesting that it could potentially act via modulating kinase activities in distinct signaling pathways (Fig.…”

A Highly Sensitive Detection Method, Phos-tag<sup>TM</sup> Affinity SDS-PAGE, Used to Analyze a Possible Substrate of CDPK-Related Protein Kinase5 in <i>Arabidopsis</i>

“…Among 10 (Figure 1(b)). Importantly, the localization of the CRK5-WDRP complex corresponds to the subcellular distribution of CRK5, which reported by Rigó et al [24].…”

“…Inactivation of CRK5 results in a root gravitropic defect and stimulates lateral root formation. CRK5 undergoes auto-activation and phosphorylates the hydrophilic T-loop of PIN2 in vitro [24].…”