Linking protein kinase CK2 and auxin transport

Studies performed in different organisms have highlighted the importance of protein kinase CK2 in cell growth and cell viability. However, the plant signaling pathways in which CK2 is involved are largely unknown. We have reported that a dominant-negative mutant of CK2 in Arabidopsis thaliana shows phenotypic traits that are typically linked to alterations in auxin-dependent processes. We demonstrated that auxin transport is, indeed, impaired in these mutant plants, and that this correlates with misexpression and mislocalization of PIN efflux transporters and of PINOID. Our data establishes a link between CK2 activity and the regulation of auxin homeostasis in plants, strongly suggesting that CK2 might be required at multiple points of the pathways regulating auxin fluxes.Key words: protein kinase CK2, root development, auxin, PIN, PINOIDThe plant hormone auxin plays critical roles in plant growth and development.¹ The most abundant natural auxin is the indol-3-acetic acid (IAA), which is synthesized in young apical tissues and then transported to the growing zones of the stem and root. The major route for long distance IAA movement is via the vascular tissue, but, additionally, a slower transport via cell-to-cell (called polar transport) is critical to generate auxin gradients within tissues. Formation of correct auxin gradients is thought to be essential for many plant developmental processes.² In recent years, the IAA transporters have been identified, establishing the molecular basis to understand how auxin transport is regulated. In particular, the identification of the family of plasma-resident PIN proteins, the members of which function as IAA efflux carriers, and the knowledge of their polar localization in the plasma membrane (PM), contributed to generate models predicting the direction of IAA fluxes.^3,4The factors that govern PIN targeting to a particular membrane domain are still not understood. It is known that PIN proteins constitutively undergo cycles of exocytosis and endocytosis to and from the PM, using distinct sorting and recycling endosome trafficking pathways.^5–7 Phosphorylation/dephosphorylation by the Ser/Thr kinase PINOID (PID) and the protein phosphatase 2A, respectively, controls PIN proteins apical/basal localization at the PM, via the GNOM-mediated vesicle trafficking system.⁸ Interestingly, PID is a member of the plant AGC kinases, and, as it happens with its mammals AGC counterparts, is activated by a membrane-associated 3-phosphoinositide-dependent kinase (PDK1).⁹ Moreover, a functional similarity between PIN polar localization in response to auxin and glucose receptor (GLUT4) asymmetrical distribution in response to insulin, has been pointed out.¹⁰ In both cases, cargo proteins (GLUT4 and PIN, respectively) are transported from endosomal vesicles to PM and the process is mediated by PDK1-activated AGC kinases.Protein kinase CK2 is a Ser/Thr kinase evolutionary conserved in eukaryotes, which plays key roles in cell survival, cell division and other cellular processes. A loss-of-function mutant of CK2 in Arabidopsis, obtained by overexpression of a CK2α-inactive subunit, confirmed the essential role of this protein kinase for plant viability.¹¹ Moreover, CK2mut plants showed a dramatic decrease of lateral root formation, inhibition of root growth and overproliferation of root hairs. We have further demonstrated that auxin transport is impaired in this plants, which is concomitant with missexpression of most of the PM-resident PIN proteins, and of PID.¹² In addition, PIN proteins accumulated in endosomal vesicles and auxin gradients were disturbed, both in roots and shoots of CK2mut plants. In particular, root columella cells were depleted of auxin, although the maximum at the quiescent center was unchanged. Starch granule staining with lugol revealed that columella cells retained their fate, although their organization and/or cell shape were clearly affected (Fig. 1).Open in a separate windowFigure 1Lugol-stained starch granules in uninduced (−Dex) and Dex-induced (+Dex) CK2mut roots. In the central part of the figure, a sketch of the main morphogenetic characteristics of mutant roots (right plantlet) as compared to wild-type roots (left plantlet) is shown. Note the shorter roots, wavy phenotype, absence of lateral roots and overproliferation of root hairs in mutant plants.Our results strongly suggest that CK2 is a regulator of auxin-dependent responses, most likely by participating in the regulation of auxin transport. Strikingly, depletion of CK2 activity inhibits some auxin-dependent physiological responses whereas it enhances others. For instance, whereas shoot phototropism was completely absent, root gravitropism was enhanced.¹² Figure 2 shows a time-course of DR5rev::GFP-derived signal after changing the gravity vector, in mutant and control Arabidopsis roots. The progressive auxin translocation to the lower side of the root after gravistimulation is more rapid and sustained in mutant than in control roots, which is likely responsible for the enhanced response to gravity found in mutant roots. Based on these results, we postulate that CK2 might act at different points of the auxin-induced regulatory pathway. As far as is known, the core module that regulates auxin transport is constituted by the protein kinase PID and a protein of the NPH3-domain family. NPH3-containing proteins play important roles in phototropic and gravitropic responses, and regulate polarity and endocytosis of PIN proteins.¹³ As has been proposed by other authors, the participation of one AGC kinase and one NPH3-like protein upstream of an ARF factor might be a common theme in response to different stimulus that are signaled by auxin.¹⁴ We propose that one of the functions of CK2 is the regulation of the activity of core proteins (Fig. 3). Mammalian AGC kinases are well known substrates of CK2 and CK2-dependent phosphorylation is critical for a full display of their activity. The PID and the NPH3-containing protein sequences contain numerous acidic-based motifs that are predicted CK2 phosphorylation sites. Moreover, according to Arabidopsis phosphoproteome databases, several members of the NPH3-containing protein family are predicted to be phosphorylated.¹⁵ In addition, we do not discard the possibility that other proteins involved in PIN transport might also be regulated by CK2-dependent phosphorylation. Experiments are in progress in our laboratory to assess the regulatory role of CK2 in auxin transport.Open in a separate windowFigure 2Time course of auxin relocation during root gravitropic response, as visualized by DR5rev::GFP fluorescence. Root pictures were taken at the indicated times after changing the direction of the gravity vector. Translocation of auxin to the lower part of the root is more rapid in Dex-induced CK2mut plants. Arrows indicate asymmetrical DR5::GFP fluorescence.Open in a separate windowFigure 3Proposed model for the role of CK2 in regulating auxin transport. The core module that regulates auxin transport (shown here as a black box) is constituted by the protein kinase PID and a protein of the NPH3-domain family. PID regulates apical-basal targeting of PIN proteins, by phosphorylating conserved Ser residues present in PIN hydrophilic loops.¹⁶ On the other hand, the family of NPH3-containing proteins regulates polarity and endocytosis of PIN proteins.¹³ There is also a functional similarity between the intracellular transport of PIN proteins and that of the glucose receptor (GLUT4),¹⁰ two processes that are signaled by AGC kinases. We propose that CK2 might be a regulator of the activity of the core proteins, by phosphorylating either the AGC kinase and/or the NPH3-containing protein. Mammalian CK2 is a known regulator of the activity of AGC kinases and other proteins participating in signaling pathways, such as in the Wnt/β-catenin signaling pathway.¹⁷