Inhibition of SnRK2 Kinases by Type 2C Protein Phosphatases.

SNF1-related protein kinase 2 s (SnRK2s) are major regulators of plant growth, development and responses to environmental stresses. Together with clade A protein phosphatases of type 2C (PP2C) and REGULATORY COMPONENTS OF ABA RECEPTOR (RCAR also known as PYRABACTIN RESISTANCE1 (PYR1) or PYR1-LIKE (PYL)) soluble abscisic acid (ABA) receptors they form the core of ABA-signaling. Clade A PP2Cs play a negative role in ABA signaling, primarily by inhibiting SnRK2 activity, through direct interaction and dephosphorylation of SnRK2s. Here, we describe two methods, which can be used for monitoring inhibition of the SnRK2 activity by PP2C phosphatases. One of them is an in vitro dephosphorylation assay using SnRK2 as the substrate followed by a classical in-gel kinase-activity assay and the other is immunocomplex kinase-activity assay, which can be applied for analysis of the SnRK2 activity in plant material.