Differentiated Inhibition Profiles of Human MAPKAPK2 Kinase

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Introduction

Due to the critical roles in intracellular communication, dysregulation of protein kinases has been implicated in as many as 400 human diseases including cancer, diabetes, heart diseases, neurological disorders and rheumatoid arthritis. Hence, individual protein kinases are important for drug target validation, protein crystal structure analysis, and drug design and screening. Promising drug leads can be screened against a panel of kinases, enabling scientists to determine the selectivity of new chemical entities. Regardless of the purpose, enzymes need to be active, highly purified, and ideally, reflect the natural post-translational modifications of the physiologically authentic protein (native state of human proteins in human cells). Currently, kinases are predominantly produced in nonhuman cells (e.g. E coli or insect cells) many of which involve protein truncation and/or in vitro activation, due to the limitation of the expression system.

HumanZyme has developed an efficient human cell-based technology, HumaXpress? for the rapid production of active recombinant human protein kinases which are full length, in vivo activated, and highly authentic. MAPKAP kinase2 (MK2) is a Ser/Thr protein kinase, which is regulated through direct phosphorylation by p38 MAP kinase. In conjunction with p38 MAP kinase, this kinase is known to be involved in many cellular processes including stress and inflammatory responses, nuclear export, gene expression regulation and cell proliferation. In this study, we demonstrate that the authentic MK2 from human cells is differentiated from the nonhuman cell version in a way that will greatly improve the speed and quality of both basic research and pharmaceutical development.

Materials and Methods

MK2- The HumanZyme MK2 kinase is fulllength containing a N-terminal GST tag. The protein was produced and activated in vivo (in human cells) in the presence of arsenite. MK2 kinase from Vendor A is a truncated form containing residues 46-400 and an Nterminal GST tag. The protein was expressed and purified from E. coli, in vitro activated with MAPK2, and repurified on Q-Sepharose FF.

Kinase Assay- The kinase assay used at Amphora Discovery detects the direct phosphorylation of a fluorescently labeled peptide substrate analog. Using Caliper’s microfluidic technology, phosphorylated product can be separated from substrate providing a quantitative determination of the ratio of the phosphorylated peptide pool.

Reaction Conditions- Unless otherwise noted these are the conditions for the MK2 kinase assay for 3 hr incubation at room temperature (25 ºC): 100 mM HEPES, pH 7.5; 1 mg/ml bovine serum albumin; 0.01% Triton X-100; 1 mM DTT; 10 mM MgCl2; 10 mM b-glycerophosphate; 10 mM sodium orthovanadate; 1 µM FAMKKLRRTLSVA- CONH2 (fluorescently labeled peptide); 100 µM ATP; 1% DMSO.

Peptide Identification- HumanZyme and Vendor A MK2 were screened for kinase activity against a collection of 192 kinase substrate peptides using the conditions described above but with 100 µM ATP.

Results

Substrate identification- Two peptides were identified as a substrate for Humanzyme MK2, KKLRRTLSVA and KIRRTLSVA. These substrates were also identified as a substrate for the Vendor A MK2. The KKLRRTLSVA sequence was used to determine the biochemical parameters of the assay.

Km Determination- The ATP Km for the kinase reaction using the identified peptide was determined using the above reaction conditions but with varying ATP concentrations. The ATP Km for the HumanZyme preparation is 5 ?0.8 mM while the Km was 15 ?2.2 ?mM for the Vendor A preparation (Fig. 1).

Inhibitor IC50- The IC50 values were determined for 13 known kinase inhibitors (Fig. 2). Reactions were as described above and incubated for 3 hr at room temperature. There was a difference in the sensitivity of select set inhibitors between the two preparations (Table 2). Both preparations were sensitive to staurosporine, AMP-PNP (a non-hydrolysable ATP analog), and K252a. The Vendor A MK2 was more sensitive to staurosporine and K252a with IC50 values 30-70 fold less than the Humanzyme MK2. In contrast, the Humanzyme MK2 was more sensitive to AMP-PNP than the Vendor A preparation, which may be a reflection of the differences in the ATP Km.

Figure 1. ATP Km

Conclusion