Ion Binding Properties of the Dehydrin ERD14 Are Dependent upon Phosphorylation

Abstract

The ERD14 protein (early response to dehydration) is a member of the dehydrin family of proteins which accumulate in response to dehydration-related environmental stresses. Here we show the Arabidopsis dehydrin, ERD14, possesses ion binding properties. ERD14 is an in vitro substrate of casein kinase II; the phosphorylation resulting both in a shift in apparent molecular mass on SDS-PAGE gels and increased calcium binding activity. The phosphorylated protein bound significantly more calcium than the nonphosphorylated protein, with a dissociation constant of 120 M and 2.86 mol of calcium bound per mol of protein. ERD14 is phosphorylated by extracts of cold-treated tissues, suggesting that the phosphorylation status of this protein might be modulated by cold-regulated kinases or phosphatases. Calcium binding properties of ERD14 purified from Arabidopsis extracts were comparable with phosphorylated Escherichia coli-expressed ERD14. Approximately 2 mol of phosphate were incorporated per mol of ERD14, indicating a minimum of two phosphorylation sites. Western blot analyses confirmed that threonine and serine are possible phosphorylation sites on ERD14. Utilizing matrix assisted laser desorption ionization-time of flight/ mass spectrometry we identified five phosphorylated peptides that were present in both in vivo and in vitro phosphorylated ERD14. Our results suggest that the polyserine (S) domain is most likely the site of phosphorylation in ERD14 responsible for the activation of calcium binding.

The K-Segment of Maize DHN1 Mediates Binding to Anionic Phospholipid Vesicles and Concomitant Structural Changes

“…Therefore, although phosphorylation does not lead to conformational changes in DHNs detectable in this study, it should not be ruled out that phosphorylation may affect lipid binding or can have some other functional role. In fact, prior studies of DHNs have shown that phosphorylation regulates nuclear localization (Jensen et al, 1998) and the binding of calcium ions (Heyen et al, 2002;Alsheikh et al, 2003;Brini et al, 2006). Taken together, even though physicochemical conditions in plant cells under dehydrative stresses are very different from those typically used to characterize biochemical properties in vitro, the results of this study suggest that the K-segments of DHN1 may be responsible for binding to negatively charged membranes in vivo and that such binding is causally related to the adoption of structure of DHN1.…”

“…For example, the Citrus unshiu CuCOR19 DHN protein binds Cu 2+ ions and scavenges reactive oxygen species, presumably to protect membranes from oxidative damage caused by water deficit (Hara et al, 2003(Hara et al, , 2005. Members of the acidic subgroup of DHNs (ERD14, ERD10, and COR47) were observed to increase Ca 2+ binding when phosphorylated in the poly-Ser motif (Alsheikh et al, 2003). It was also recognized that acidic residues of DHNs are involved in ion binding (Alsheikh et al, 2005).…”

“…On the basis of compositional and biophysical properties and their link to abiotic stresses, several functions of DHNs have been proposed, including ion sequestration (Roberts et al, 1993), water retention (McCubbin et al, 1985), and stabilization of membranes or proteins (Close, 1996(Close, , 1997. Observations from in vitro experiments include DHN binding to lipid vesicles (Koag et al, 2003;Kovacs et al, 2008) or metals (Svensson et al, 2000;Heyen et al, 2002;Kruger et al, 2002;Alsheikh et al, 2003;Hara et al, 2005), protection of membrane lipid against peroxidation (Hara et al, 2003), retention of hydration or ion sequestration (Bokor et al, 2005;Tompa et al, 2006), and chaperone activity against the heat-induced inactivation and aggregation of various proteins (Kovacs et al, 2008).…”

Tunable Membrane Binding of the Intrinsically Disordered Dehydrin Lti30, a Cold-Induced Plant Stress Protein

“…For an unambiguous identification of the sequence segments of Lti30 that serve to assemble the vesicles, we added flanking His residues to the canonical K-segments in the form of the synthetic peptide HHEKKGM-TEKVMEKIKEQLPGHH. Addition of this isolated His-flanked K-segment to PC:PG vesicles at pH 4.3 induces aggregation indistinguishable from that of the full-length protein (see Phosphorylation of the dehydrins is observed to take place both in vivo and in vitro, indicating a role in functional regulation in stressed plant cells (Alsheikh et al, 2003;Jiang and Wang, 2004;Rö hrig et al, 2006;Brini et al, 2007). The sequence algorithm Netphos (Expasy) predicts that Lti30 is specifically phosphorylated by protein kinase C (PKC) at nine different positions, several of which are in the K-segments (Figure 1).…”

The Importance of Size and Disorder in the Cryoprotective Effects of Dehydrins

“…Dehydrins are thought to be involved in dehydration protection, since their transcription and translation are increased during dehydration, and a correlation exists between drought tolerance and the amount of dehydrin present. In vitro, dehydrins have been shown to protect enzymes from freeze-thaw damage (Lin and Thomashow, 1992;Kazuoka and Oeda, 1994;Momma et al, 2003;Goyal et al, 2005;Hughes and Graether, 2011) and heat denaturation (Kovacs et al, 2008), interact with and protect membranes from cold and dehydrative stresses (Rahman et al, 2010;, and bind water (Tompa et al, 2006), ions (Alsheikh et al, 2003), and nucleic acids (Hara et al, 2009). Dehydrins have also been suggested to prevent the growth of ice crystals by functioning in a manner similar to antifreeze proteins (AFPs; Wisniewski et al, 1999;Simpson et al, 2005).…”