Differentiated Inhibition Profiles of Human p38a
Kinase from Human Cells vs Non-human Cells
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. p38a, a
member of the MAP kinase family, is a critical
mediator of the production of inflammatory
cytokines. In fact, p38 was originally identified
as the molecular target of a class of small
chemicals with promising anti-inflammatory
and anti-rheumatic properties. In this study,
we demonstrate that p38a from human
cells is differentiated from non-human cell
versions in a way that will greatly improve the
speed and quality of both basic research and
pharmaceutical development.
Materials and Methods
HumanZyme p38a - The p38a kinase was
produced and activated in vivo (in human
cells) in the presence of arsenite. p38a
kinases from Amphora Discovery and Vendor
A were expressed and purified from E. coli, b
activated by MKK6, and repurified.
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 p38a
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 µM?glycerophosphate; 10
µM sodium orthovanadate; 1 µM FAMIPTSPITTTYFFFKKK-
CONH2 (fluorescently
labeled peptide); 200 µM ATP; 1% DMSO.
Peptide Identification - HumanZyme p38a
was screened for kinase activity against a
collection of 192 kinase substrate peptides using the conditions described above but
with 100µM ATP and 0.1 µg/ml Humanzyme
p38a.
Results
Purity and activity of HumanZyme p38a -
SDS PAGE analysis shows that the enzyme
is pure with a dominant band of 60 kD and
minor band of endogenous human GST of
23 kD. This was confirmed by MS analysis.
No other contaminant proteins were found.
The specific activity was 125U/mg. One unit
of kinase activity is defined as 1nmole ATP
consumed in the kinase reaction assay in the
presence of 0.5mg/ml myelin basic protein
(MBP) substrate per minute at 25°C with a
final ATP concentration of 100µM.
Substrate identification - Only one peptide
was identified as a substrate for HumanZyme
p38a (IPTSPITTTYFFFKKK-CONH2). This
substrate was also identified as a substrate
for the Amphora and Vendor A p38a. Thus,
this 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 109 ?2µM while the Km was 212 ?6 µM
for the Amphora preparation (Fig. 1).The Km
of 120 µM was found with Vendor A enzyme.
Subsequent reactions were executed at two
times the Km or 200µM.
Reaction Rate - The rate of the reaction
was determined at 25 ºC over a 3 hr period
at several concentrations of p38a. The rate
of the reaction with HumanZyme p38a had
nearly a sixty minute lag in phosphorylation.
The Amphora preparation had a comparatively
marginal lag likely due to the higher specific
Figure 1. ATP Km
activity of the preparation. For a 3 hr. reaction at
room temperature the specific activity was 3.6
µg/ml and 0.5 µg/ml for the HumanZyme and
Amphora p38a preparations respectively. The
activity of Vendor A enzyme was comparable
to that of HumanZyme.
Inhibitor IC50 - The IC50 values were
determined for 14 known kinase inhibitors
(Fig. 2). Reactions were as described above
and incubated for 3 hr. at room temperature.
While the IC50 values for SB-202190 (the
p38a selective inhibitor) for both p38a
preparations were similar (0.02 uM and 0.03
µM respectively), there is clearly a difference
in the sensitivity of the inhibitors between
the two preparations (Fig. 2; Table 1). The
Amphora preparation was only sensitive to
AMP-PNP (a non-hydrolysable ATP analog).
Yet, the protein was 7-fold less sensitive than
the HumanZyme enzyme, which is consistent
with its higher Km. Humanzyme p38a, on
the other hand, had measurable IC50 values
against staurosporine, K252a, Ro 31-8220,
KT5720, and SB-202190. The inhibition
profile of Vendor A kinase is comparable to
that of Amphora.
Table 1. Inhibitor IC50 Values
Figure 2. Inhibitor IC50 Curves
Conclusion
The current study demonstrates that the
properties of the human protein kinase
p38a produced in human cells are clearly
differentiated from versions of the same kinase
that were produced in non-human cell systems.
Given the high cost and lengthy development
of drugs, it would be highly justified to use
human kinases with high authenticity for drug
screening as well as kinase profiling.
This study was conducted in collaboration
with Amphora (amphoracorp.com)
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