Calcium/calmodulin-regulated receptor-like kinase CRLK1 interacts with MEKK1 in plants

Recently we reported that CRLK1, a novel calcium/calmodulin-regulated receptor-like kinase plays an important role in regulating plant cold tolerance. Calcium/calmodulin binds to CRLK1 and upregulates its activity. Gene knockout and complementation studies revealed that CRLK1 is a positive regulator of plant response to chilling and freezing temperatures. Here we show that MEKK1, a member of MAP kinase kinase kinase family, interacts with CRLK1 both in vitro and in planta. The cold triggered MAP kinase activation in wild-type plants was abolished in crlk1 knockout mutants. Similarly, the cold induced expression levels of genes involved in MAP kinase signaling are also altered in crlk1 mutants. These results suggest that calcium/calmodulinregulated CRLK1 modulates cold acclimation through MAP kinase cascade in plants.Key words: calcium, calmodulin, cold stress, MAPK, Arabidopsis, protein phosphorylationCalcium, a universal second messenger in eukaryotic cells, mediates changes in external and internal signals leading to the physiological responses.^1–4 Calcium/calmodulin (Ca²⁺/CaM)-dependent protein kinases (CaMKs) are very important players in calcium/calmodulin mediated signaling in mammalian cells.⁵ In plants, Ca²⁺/CaM-dependent protein phosphorylation was observed more than 25 years ago.⁶ Several calmodulin-regulated protein kinases have been identified and characterized.^7,8 For example, plants have a unique chimeric Ca²⁺/CaM-dependent protein kinase (CCaMK), which exhibits Ca²⁺-dependent autophosphorylation and Ca²⁺/CaM-dependent substrate phosphorylation.⁹ CCaMK is required for bacterial and fungal symbioses in plants.^10–12 Recently, we characterized a novel plant-specific calcium/CaM-regulated receptor-like kinase, CRLK1.¹³ Ca²⁺/CaM binds to CRLK1 and stimulates its kinase activity. Functional studies with CRLK1 indicate that CRLK1 acts as a positive regulator in plant response to chilling and freezing temperatures. To further define the CRLK1-mediated signal pathway, we isolated CRLK1 interacting proteins by co-immunoprecipitation using an anti-CRLK1 antibody. Since cold increases the amount of CRLK1 protein, wildtype plants (WT) were treated at 4°C for 1 hr before co-immunoprecipitation. The resulting CRLK1 immunocomplex was separated by SDS-PAGE. We observed several bands of different sizes only in the wild-type but not in the crlk1 knockout mutant plants (Fig. 1A). Furthermore, the intensity of these bands increased upon cold treatment, suggesting that they are the putative partners or associated proteins of the CRLK1 immunocomplex.Open in a separate windowFigure 1CRLK1 Interacts with MEKK1. (A) One-dimension SDS-PAGE of anti-CRLK1 immunocomplexes from 3-week-old WT or crlk1 plants with or without cold treatment. One mg of total protein was used for immunoprecipitation. (B) A list of putative CRLK1-interacting proteins determined by MALDI-TOF-MS analysis. (C) CRLK1 interacts with MEKK1 as shown by GST pull-down assay. (D) BiFC analysis show that CR LK associates with MEKK1 in vivo. Upper row shows that CRLK and MEKK1 associate both on cell membrane and in endosomes. The middle and last rows are controls. Bar = 10 µm.To determine the identities of these proteins, mass spectrometric analysis was performed with the total immunocomplex.¹⁴ In addition to CRLK1, there were 12 other proteins which matched the Arabidopsis database. Several of them appeared in the pull-down complex from WT, but not from crlk1 mutants. These putative interacting proteins included MEKK1, another unknown protein kinase, a type 2C phosphatase and CaM (Fig. 1B). MEKK1 is one of the 60 putative MAPKKKs in the Arabidopsis genome, and sits on the top of mitogen-activated protein kinase (MAPK) cascade. The MAPK signaling consists of a cascade of three consecutively acting protein kinases, a MAP kinase kinase kinase (MAPKKK), a MAP kinase kinase (MAPKK) and a MAP kinase (MAPK). Plants possess multiple MAPKKKs, MAPKKs and MAPKs, which respond to different upstream signals and activate distinct downstream pathways.^15–17 The specific MAPK module responding to lower temperature has been determined in Arabidopsis.^18,19 MEKK1, a member of MAPKKKs, specifically interacts and phosphorylates MKK2 and regulates COR genes expression in response to cold stress.¹⁹ MEKK1 has been shown to play a role in mediating reactive oxygen species homeostasis.^20,21 Therefore we selected MEKK1 from the putative CRLK1 partners for further studies.