Enhancement of ASK1 kinase activity by MPK38. A,
stimulation of ASK1 kinase activity by MPK38. 293T cells were transiently
transfected with HA-ASK1 in the presence or absence of GST-MPK38. Cell lysates
were subjected to immunoprecipitation (IP) with an anti-HA antibody,
and the HA immunoprecipitates were analyzed for ASK1 kinase activity by an
in vitro kinase assay using recombinant MKK6(K82A) as a substrate.
The circled P-MKK6(K82A) indicates phosphorylated MKK6(K82A). The
same blot was stripped and re-probed with an anti-HA antibody to determine the
expression level of immunoprecipitated ASK1 (left, 2nd panel), and
the expression level of GST-MPK38 in total cell lysates was examined by
immunoblot analysis using an anti-GST antibody. (left, 3rd panel).
The presence of equivalent amounts of substrate was verified by immunoblotting
with an anti-GST antibody (left, bottom panel). 5 μg of
recombinant ASK1-K (re.ASK1-K) or wild-type ASK1 (re.ASK1)
proteins were incubated at room temperature for 1 h with the indicated amount
of recombinant MPK38 (re.MPK38) in 50 μl of 25 mm HEPES
buffer, pH 7.4, and then analyzed for ASK1 kinase activity by an in
vitro kinase assay using recombinant MKK6(K82A) as a substrate
(middle and right). WB, Western blot. B,
requirement of MPK38 activity for MPK38-induced stimulation of ASK1 kinase
activity. After 48 h of transfection with the indicated expression vectors,
293T cell lysates were subjected to immunoprecipitation with an anti-HA
antibody and then analyzed by an in vitro kinase assay with
recombinant MKK6(K82A) substrate (top panel). ASK1 Thr838
phosphorylation in the HA immunoprecipitates was determined by immunoblot
analysis using an anti-phospho-ASK1(T845) antibody (2nd
panel). The expression levels of immunoprecipitated ASK1 and wild-type
and kinase-dead MPK38 in total cell lysates were analyzed using anti-HA and
anti-GST antibodies, respectively (3rd and 4th panels). The
relative level of ASK1 kinase activity was quantitated by densitometric
analyses, and fold increase relative to control expressing ASK1 or ASK1-K
alone was calculated. re., recombinant.