New Candidate Targets of AMP-Activated Protein Kinase in Murine Brain Revealed by a Novel Multidimensional Substrate-Screen for Protein Kinases

AMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine kinase that is involved in the
maintenance of energy homeostasis and recovery from metabolic stresses both at the cellular and
whole body level. AMPK is found in all tissues examined so far, and a number of downstream targets
have been identified. Recent work suggests that AMPK has specialized functions in the brain, such as
involvement in appetite control. Nevertheless, brain-specific substrates of AMPK are unknown. Here,
we performed a proteomic in vitro screen to identify new putative AMPK targets in brain. Prefractionation
of murine brain lysates by liquid chromatography, utilizing four different, serially connected columns
with different chemistries was found to be superior to a single column method. A pilot screen involving
incubation of small volumes of individual fractions with radiolabeled ATP in the presence or absence
of active AMPK, followed by one-dimensional SDS-PAGE and autoradiography, revealed the presence
of potential AMPK substrates in a number of different fractions. On the basis of these results, several
kinase assays were repeated with selected fractions on a preparative scale. Following separation of
the radiolabeled proteins by two-dimensional electrophoresis and comparison of samples with or
without added AMPK by differential autoradiography, 53 AMPK-specific phospho-spots were detected
and excised. Thereof, 26 unique proteins were identified by mass spectrometry and were considered
as new potential downstream targets of AMPK. Kinase assays with 14 highly purified candidate substrate
proteins confirmed that at least 12 were direct targets of AMPK in vitro. Although the physiological
consequences of these phosphorylation events remain to be established, hypotheses concerning the
most intriguing potential targets of AMPK that have been identified by this search are discussed herein.
Our data suggests that signaling by AMPK in brain is likely to be involved in the regulation of pathways
that have not yet been linked to this kinase.
Keywords: AMP-activated protein kinase • phosphorylation • multidimensional separation • liquid chromatography
• two-dimensional electrophoresis • phosphoproteomics • MALDI • mass spectrometry • multidimensional substrate-screen for protein kinases • MudSeek