AMPK Methods and Protocols

3.3 Modification
of the Protocol
for Postharvest
Increase or Decrease
of AMPK Activity

We find that recombinant AMPK extracted from COS7 cells, incu-

bated in high-glucose DMEM, has measurable kinase activity in the

low nmol.min
^1 .mg
^1 enzyme range (Table2). For drug screening

purposes, it is often useful to obtain AMPK preparations with either

higher activity (e.g., amenable to inhibitor screening [10]) or neg-

ligible activity (e.g., amenable to screening for AMP synergism

[11]). Since AMPK activity is directly related to phosphorylation

of Thr-172 in the activation loop, modulation of AMPK activity can

be conveniently performed by treatment with CaMKK2 (Thr-172

phosphorylation) or lambda (λ) phosphatase (phospho-Thr-172

dephosphorylation) (Fig.2). Treatments can be conveniently per-

formed while AMPK is immobilized on glutathione agarose.

1. CaMKK2 can be purchased commercially, but we find expres-
   sion of recombinant enzyme in mammalian cells a more conve-
   nient and economical source in our laboratory. The method is
   identical tosteps 1– 3 in Subheading3.1, with the exception
   that 3μg of pcDNA3-CaMKK2-FLAG DNA is transfected per
   10 cm dish and cell lysates are harvested in ice-cold nonreduc-
   ing cell lysis buffer (seeNote 16).


2. For the purification, dispense anti-FLAG M2 affinity agarose
   gel (~20μl of a 50% slurry/0.5 ml cell lysate) into a 1.5 ml
   Eppendorf tube, and pellet by centrifugation at 3,350gfor
   3 min.

Table 2
Expected specific activities of recombinant AMPK complexes expressed in COS7 cells. Fold increases
in enzyme activity in the presence of 200μM AMP (þAMP) or CaMKK2 treatment, relative to basal,
are indicated

AMPK complex Basal activity (nmol.min
^1 .mg
^1 enzyme)

Fold stimulation

þAMP CaMKK2-treated

α 1 β 1 γ 1 100–200 2–3 20–30

α 1 β 1 γ 2 100–200 2–3 20–30

α 1 β 1 γ 3 100–200 1.5 20–30

pT172

1

-ppase

CaMKK2

+ - -

Fig. 2 Postharvest treatments of glutathione-agarose immobilized AMPK

α 1 β 1 γ1. CaMKK2 or λ-phosphatase treatments either increase, or reduce,

α-Thr-172 phosphorylation, respectively

Production in Mammalian Cells 165