An ATP analog-sensitive version of the tomato cell death suppressor protein kinase Adi3 for use in substrate identification

Abstract

Adi3 is a protein kinase from tomato that functions as a cell death suppressor and its substrates are not well defined. As a step toward identifying Adi3 substrates we developed an ATP analog-sensitive version of Adi3 in which the ATP-binding pocket is mutated to allow use of bulky ATP analogs. Met385 was identified as the “gatekeeper” residue and the M385G mutation allows for the use of two bulky ATP analogs. Adi3^M385G can also specifically utilize N⁶-benzyl-ATP to phosphorylate a known substrate and provides a tool for identifying Adi3 substrates.

Introduction

The genetically encoded process of programmed cell death (PCD) is known to exist in many organisms including multi- and single cell eukaryotes [1], [2] and even in some bacteria [3]. PCD is often associated with the successful completion of developmental processes, resistance to pathogens, and responses to abiotic stress [1], [4], [5]. Despite the importance of PCD, plant genes that regulate PCD have been difficult to identify [1], [6], [7]. However, in recent years many genes that encode proteins involved in signaling pathways linked to PCD control have been identified [8], [9], [10], [11], [12], [13], [14], [15]. But, the pathways associated with these proteins have yet to be fully elucidated.

In tomato, the Ser/Thr protein kinase Adi3 has been identified as a suppressor of PCD and is connected to the control of cell death during the resistance to Pseudomonas syringae pv. tomato, the causative agent of bacterial speck disease. Our previous studies have identified portions of the Adi3 signaling pathway including the upstream kinase 3-phosphoinositide-dependent protein kinase-1 (Pdk1), which phosphorylates Adi3 at Ser539, and the mitogen-activated protein kinase MAPKKKα that acts in a downstream parallel pathway [16], [17]. We have also identified Adi3 interacting proteins using a yeast two-hybrid screen, but only identified one substrate for Adi3 [18], possibly due to the transient nature of kinase–substrate interactions, which is not ideal for yeast two-hybrid screens [19].

In order to isolate additional Adi3 substrates, we have developed an ATP analog-sensitive (as-) form of Adi3 (as-Adi3). In this approach, an as-kinase is produced by mutating a bulky, or “gatekeeper”, amino acid within the ATP-binding pocket to a less bulky amino acid such as Ala or Gly [20], [21]. This creates a larger ATP-binding pocket and allows for the specific use of bulky forms of ATP with N⁶ substitutions such as N⁶-benzyl-ATP or N⁶-phenylethyl-ATP [20], [21] (Fig. 1). Only the as-kinase can utilize the bulky ATP analogs and thus, can be used to specifically identify phosphorylation substrates from cell extracts. The production and use of as-kinases have been successfully used on dozens of kinases from yeast and mammalian systems [20], [22], but this technology has rarely been used on plant kinases [22], [23], [24], [25]. Here we report the development of an as-Adi3 and show that it can specifically utilize N⁶-benzyl-ATP to phosphorylate a known Adi3 substrate.