Background

Extracellular signal-regulated kinase 3 (ERK3) is an atypical mitogen-activated protein kinase (MAPK) (Coulombe and Meloche, 2007). Here we describe an in vitro kinase assay in which ERK3 transfers radiolabeled gamma-phosphate from [γ-³²P]-ATP to a purified protein substrate. Excess radiolabeled ATP is then separated from the radiolabeled substrate by gel electrophoresis. The amount of phosphorylated substrate can be quantified by autoradiography, phosphorimaging, or liquid scintillation counting techniques. Radioactive kinase assay provides a direct measurement of kinase activity. It is sensitive, quick, inexpensive, and considered as the 'gold standard' for quantification of protein kinase activity. The major limitations of radioactive kinase assays are the hazards of handling radiolabeled isotopes and unsuitability of this assay format for large scale high-throughput screening.

This protocol describes a direct kinase assay for ERK3 using myelin basic protein (MBP) or a fragment of steroid receptor coactivator-3 (SRC-3) as substrates. MBP is a non-specific substrate for several kinases including members of MAPK family (Haubrich and Swinney, 2016). SRC-3 was shown to interact with ERK3 and is phosphorylated by ERK3 on its Ser⁸⁵⁷ residue within the CBP-interacting domain (CID) (Long et al., 2012). Hence, SRC3-CID fragment, which comprises amino acids 841-1080, is used as a substrate in this assay.

Another substrate for ERK3 that has been well-characterized and validated to be physiologically relevant is MAPK-activated protein kinase 5 (MK5) (Schumacher et al., 2004, Seternes et al., 2004). ERK3 phosphorylates MK5 at Thr¹⁸², leading to MK5 activation. Since MK5 itself is also a kinase, the activity of ERK3 towards MK5 has been determined by a coupled kinase assay in which the phosphorylation of peptide or protein substrate for MK5 is measured in the presence of both ERK3 and MK5 (Schumacher et al., 2004, Seternes et al., 2004).

The in vitro kinase assay described here has been previously used to identify novel ERK3 substrates (Bian et al., 2016), to determine the effect of mutations on the kinase activity of ERK3 (Alsaran et al., 2017, Elkhadragy et al., 2018), and to compare autophosphorylation of wild type or mutant ERK3 (Elkhadragy et al., 2018). In these studies, ERK3 protein was expressed and purified from bacteria, Sf9 insect cells, or mammalian cells. HA-tagged ERK3 expressed and immunoprecipitated from mammalian 293T cells showed greater in vitro kinase activity as compared to recombinant His-tagged ERK3 purified from E. coli (Elkhadragy et al., 2018), possibly because of the greater extent of post-translational modifications or the presence of interacting partners in mammalian cells. Selection of the method for purifying ERK3 protein to be used in a kinase assay has to be based on the purpose and specific considerations of the experiment to be conducted.