Recent Advances in Substrate Identification of Protein Kinases in Plants and Their Role in Stress Management

Abstract

Protein phosphorylation-dephosphorylation is a well-known regulatory mechanism in biological systems and has become one of the significant means of protein function regulation, modulating most of the biological processes. Protein kinases play vital role in numerous cellular processes. Kinases transduce external signal into responses such as growth, immunity and stress tolerance through phosphorylation of their target proteins. In order to understand these cellular processes at the molecular level, one needs to be aware of the different substrates targeted by protein kinases. Advancement in tools and techniques has bestowed practice of multiple approaches that enable target identification of kinases. However, so far none of the methodologies has been proved to be as good as a panacea for the substrate identification. In this review, the recent advances that have been made in the identifications of putative substrates and the implications of these kinases and their substrates in stress management are discussed.

Keywords: Kinase; Phosphorylation; Post-translational modification; Signal transduction; Stress management; Substrate identification.

Fig. (1)

Substrates of kinases screened and validated by Y2H (yeast two-hybrid), PRs (protein microarrays), ImPpT (immunoprecipitation), FRET (Fluorescence Resonance Energy Transfer) and BiFC (Bimolecular fluorescence complementation assay). The green oval shows the kinases, rectangular boxes indicate techniques used for screening of putative substrates followed by its further confirmation while pink oval represents substrates identified. Zhu et al. in 2001[85] used Protein microarray for the identification of substrates for yeast kinases. Immunoprecipitation technique helped to validate interaction of v-Crk SH2 domain and demonstration of paxillin phosphorylation by GST fusion immuno-precipitation [29]. FRET aided in reporting the in-vivo interactions of the MAPK from Arabidopsis thaliana - MPK6 with an Ethylene Responsive Factor104 (ERF104). Identification of putative substrate (transcription factor) of MPK6 by screening through yeast-two hybrid, followed by validation of interaction in-vivo by FRET [48]. Ito and group confirmed the interaction of NtCDPK with RSG (REPRESSION OF SHOOT GROWTH- a transcription factor involved in gibberellin homeostasis) by BiFC by fusing RSG and NtCDPK with YFPn and YFPc, respectively thus confirming their interaction. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)

Fig. (2)

A general outline of abiotic stress responses and implication of kinases therein. Responses against abiotic stresses such as flooding, cold, salinity, nutritional deficiency and others bring about many plant kinases in signaling map. These kinases impart adaptation and tolerance against these stresses through phosphorylating the downstream target proteins which includes transcription factors (TFs), enzymes etc.

Fig. (3)

A general outline of biotic stress responses and implications of MAP kinases therein. Activation of plant Mitogen-Activated Protein Kinase (MAPK) cascade is one of the earliest signaling events in PTI (Pathogen-Associated Molecular Patterns (PAMPs) triggered immunity) and ETI (Effector Triggered Immunity). Phosphorylation-the most common post-translational modification is mediated by these MAPKs and phosphatases, which fine-tune these signaling events. The phosphorylation of target proteins, which include transcription factors, enzymes etc. promotes synthesis/or signaling of defense hormones, activation of defense genes, synthesis of nitric oxide (NO) to mediate stomatal closure, hypersensitive responses (HR)-like cell death and other defense responses.