Activation of AMP-activated protein kinase (AMPK) through inhibiting interaction with prohibitins.

5'-Adenosine monophosphate-activated protein kinase (AMPK) is a potential therapeutic target for various medical conditions. We here identify a small-molecule compound (RX-375) that activates AMPK and inhibits fatty acid synthesis in cultured human hepatocytes. RX-375 does not bind to AMPK but interacts with prohibitins (PHB1 and PHB2), which were found to form a complex with AMPK. RX-375 induced dissociation of this complex, and PHBs knockdown resulted in AMPK activation, in the cultured cells. Administration of RX-375 to obese mice activated AMPK and ameliorated steatosis in the liver. High-throughput screening based on disruption of the AMPK-PHB interaction identified a second small-molecule compound that activates AMPK, confirming the importance of this interaction in the regulation of AMPK. Our results thus indicate that PHBs are previously unrecognized negative regulators of AMPK, and that compounds that prevent the AMPK-PHB interaction constitute a class of AMPK activator.

Relative resistance of Cdk5-phosphorylated CRMP2 to dephosphorylation.

Collapsin response mediator protein 2 (CRMP2) binds to microtubules and regulates axon outgrowth in neurons. This action is regulated by sequential phosphorylation by the kinases cyclin-dependent kinase 5 (Cdk5) and glycogen synthase kinase 3 (GSK3) at sites that are hyperphosphorylated in Alzheimer disease. The increased phosphorylation in Alzheimer disease could be due to increases in Cdk5 and/or GSK3 activity or, alternatively, through decreased activity of a CRMP phosphatase. Here we establish that dephosphorylation of CRMP2 at the residues targeted by GSK3 (Ser-518/Thr-514/Thr-509) is carried out by a protein phosphatase 1 family member in vitro, in neuroblastoma cells, and primary cortical neurons. Inhibition of GSK3 activity using insulin-like growth factor-1 or the highly selective inhibitor CT99021 causes rapid dephosphorylation of CRMP2 at these sites. In contrast, pharmacological inhibition of Cdk5 using purvalanol results in only a gradual and incomplete dephosphorylation of CRMP2 at the site targeted by Cdk5 (Ser-522), suggesting a distinct phosphatase targets this residue. A direct comparison of dephosphorylation at the Cdk5 versus GSK3 sites in vitro shows that the Cdk5 site is comparatively resistant to phosphatase treatment. The presence of the peptidyl-prolyl isomerase enzyme, Pin1, does not affect dephosphorylation of Ser-522 in vitro, in cells, or in Pin1 transgenic mice. Instead, the relatively high resistance of this site to phosphatase treatment is at least in part due to the presence of basic residues located nearby. Similar sequences in Tau are also highly resistant to phosphatase treatment. We propose that relative resistance to phosphatases might be a common feature of Cdk5 substrates and could contribute to the hyperphosphorylation of CRMP2 and Tau observed in Alzheimer disease.

Novel procedure to investigate the effect of phosphorylation on protein complex formation in vitro and in cells.

The identification of phosphorylation state-dependent interacting proteins provides clues as to the function of the phosphorylation. Techniques such as yeast two hybrid and co-immunoprecipitation do not employ a single species of fully phosphorylated proteins. This is a particular problem for substrates of glycogen synthase kinase-3 (GSK3), where multiple Ser/Thr residues can be targeted, almost always subsequent to a priming phosphorylation by an alternative kinase. We previously identified the brain enriched collapsin response mediator proteins (CRMP2 and CRMP4) as physiological substrates of GSK3. Cdk5 phosphorylates CRMP2 at Ser522, priming for subsequent phosphorylation at three residues by GSK3 in vitro and in vivo. It is clear that phosphorylation of CRMP2 influences axonal growth; however, the molecular processes underlying this action are not fully established. In addition, the role of phosphorylation in other actions of CRMPs has not been elucidated. We developed a novel procedure to isolate CRMP2 and CRMP4 fully phosphorylated at four sites, namely, Ser522 (by CDK5), Ser518, Thr514, and Thr509 (by GSK3). These phosphoproteins were then used to identify binding partners in rat brain lysates in direct comparison with the non-phosphorylated isoforms. We validated the approach by confirming that a previously reported interaction with tubulin-beta is regulated by phosphorylation. We also show that CRMPs (CRMP1, CRMP2, and CRMP4) form heteromers and found that these complexes may also be regulated by phosphorylation. We identified DYRK and Pin1 as novel CRMP4 binding proteins with DYRK interacting preferentially with dephospho-CRMP4 and Pin1 with phospho-CRMP4. Finally, we used this approach to identify the mitochondrial protein ANT as a novel CRMP2 and CRMP4 binding protein. We believe that this approach could be applied generally to the study of phosphorylation-dependent interactions.