Phosphorylation of Arabidopsis thaliana MEKK1 via Ca2+ signaling as a part of the cold stress response

Abstract

The Arabidopsis mitogen activated protein kinase kinase kinase (MEKK1) plays an important role in stress signaling. However, little is known about the upstream pathways of MEKK1. This report describes the regulation of MEKK1 activity during cold signaling. Immunoprecipitated MEKK1 from cold-treated Arabidopsis seedlings showed elevated kinase activity towards mitogen activated protein kinase kinase2 (MKK2), one of the candidate MEKK1 substrates. To clarify how MEKK1 becomes active in response to cold stress signaling, MEKK1 phosphorylation was monitored by an enzyme extracted from the seedlings grown under cold stress with or without EGTA. MEKK1 was phosphorylated after cold stress, but EGTA inhibited the phosphorylation. MKK2 was also phosphorylated by the same extract, but only when EGTA was absent. These results suggested that Ca(2+) signaling occurred upstream of the MEKK1-MKK2 pathway. Full-length MEKK1 showed almost no activity but MEKK1 without the N-terminal region (MEKK1 KD) that retained the kinase domain had a strong ability to phosphorylate MKK2, demonstrating the inhibitory role of the N-terminal region of MEKK1. In addition, MEKK1 was phosphorylated by calcium/calmodulin-regulated receptor-like kinase (CRLK1), which suggested that CRLK1 is one of candidates located upstream of MEKK1.

MAP Kinase Cascades Regulate the Cold Response by Modulating ICE1 Protein Stability

“…Under cold treatment, the phosphorylation levels of MPK3, MPK4, and MPK6 were significantly increased (Figure 1A), whereas the phosphorylation of MPK8 was significantly decreased (p-value<0.01) (Table S1). These results are consistent with previous studies (Furuya et al, 2013; Teige et al, 2004), which showed that MEKK1, MKK2, MPK4, and MPK6 are activated after cold treatment. Here, our method detected MPK3 as another MAPK responsive to cold conditions.…”

“…In summary, previous studies reported that a MEKK1-MKK2-MPK4/MPK6 pathway is activated after cold treatment (Teige et al, 2004), and CRLK1, which is stimulated by binding to calmodulin and calcium, a second messenger in plants under cold stress, physically associates with and phosphorylates MEKK1 (Furuya et al, 2013; Yang et al, 2010a, 2010b). In this study, we found that MKK2 is the upstream kinase of MPK4 but not MPK3/6 in the cold signaling pathway.…”

“…Previous studies showed that the MEKK1-MKK2-MPK4 cascade positively regulates cold response (Furuya et al, 2013; Teige et al, 2004). In this study, we found that indeed the expression levels of CBF genes were substantially down-regulated in mpk4 mutant plants (Figures 4A–4C), and the mpk4 mutant was hypersensitive to freezing (Figure 4D).…”

“…Previous studies showed that CRLK1, a calcium/calmodulin-regulated receptor-like kinase, regulates cold-responsive gene expression and freezing tolerance (Yang et al, 2010a), likely through direct interaction and phosphorylation of MEKK1 (Furuya et al, 2013; Yang et al, 2010b). CRLK2 is a paralog of CRLK1 in Arabidopsis .…”

“…Previous studies suggested that a MEKK1-MKK2-MPK4/MPK6 cascade is involved in the positive regulation of cold response and freezing tolerance (Teige et al, 2004). Low temperatures induce the kinase activity of MEKK1, which subsequently phosphorylates MKK2 (Furuya et al, 2013). Activated MKK2 phosphorylates MPK4 and MPK6 (Teige et al, 2004), which then presumably regulate downstream components to adjust the cellular status to adapt to freezing conditions.…”