The LKB1-AMPK pathway: metabolism and growth control in tumour suppression

Abstract

In the past decade, studies of the human tumour suppressor LKB1 have uncovered a novel signalling pathway that links cell metabolism to growth control and cell polarity. LKB1 encodes a serine-threonine kinase that directly phosphorylates and activates AMPK, a central metabolic sensor. AMPK regulates lipid, cholesterol and glucose metabolism in specialized metabolic tissues, such as liver, muscle and adipose tissue. This function has made AMPK a key therapeutic target in patients with diabetes. The connection of AMPK with several tumour suppressors suggests that therapeutic manipulation of this pathway using established diabetes drugs warrants further investigation in patients with cancer.

Figures

Figure 1. Schematic of the proteins in the LKB1 and AMPK kinase complexes

Both LKB1 and AMPK exist in heterotrimeric protein complexes. Inactivating mutations in LKB1 underlie the inherited cancer disorder Peutz-Jeghers Syndrome. Most mutations affect the function of the kinase domain, indicating that the tumor suppressor function of LKB1 requires its kinase activity. In addition to deletions or frameshifts, several missense mutations have been found and most cluster to the kinase domain resulting in loss of kinase activity. A handful of mutations lie outside the kinase domain and some of these have been shown to result in decreased kinase activity due to disruption of protein-protein interactions between LKB1 and its regulatory subunits STRAD (STE20-related adapter protein) and Mo25, which appear to be necessary for its kinase activity. Together, the genetic evidence indicate that the tumor suppressor function of LKB1 requires its kinase activity. While there is a single LKB1 gene in mammals, two STRAD and two Mo25 family members exist and mutations in STRADα underlie the development of an inherited epileptic disorder. There are two known splice forms of LKB1 differing in the very C-terminal amino acids, , and evidence suggests STRAD proteins undergo extensive alternative splicing as well. Like LKB1, AMPK is composed of a catalytic subunit (α) and two regulatory subunits. The beta subunits contain a conserved glycogen binding domain which also modulates AMPK activity. The gamma subunits contain a series of tandem repeats of crystathionine-β-synthase (CBS) domains to which molecules of AMP directly bind as revealed in recent X-ray crystallography studies. Binding of AMP to AMPKγ is thought to promote phosphorylation of the critical activation loop threonine (Thr172) in AMPKα, which is required for AMPK activity, largely through suppression of phosphatase activity towards Thr172. Mutation of some of these AMP-binding pockets in the AMPKγ2 gene lead to hypertrophic cardiomyopathy that is associated with Wolff-Parkinson-White syndrome.

Figure 2. LKB1-dependent signaling

LKB1 in complex with its two regulatory subunits STRAD and Mo25 directly phosphorylates and activates a family of 14 AMPK-related kinases. These kinases in turn directly phosphorylate a number of downstream substrates to mediate effects on cell polarity, metabolism, and growth control. All well-established substrates of AMPK and its related family members are shown, and those for which further in vivo data is needed are shown with a question mark. It is important to note that many of the known substrates are expressed in a tissue-specific manner and may not explain ubiquitous effects of LKB1 and its downstream kinases in all cell types. Bottom: The sequences flanking the best-characterized phosphorylation site in each substrate with those residues selected for from in vitro peptide library and alanaine scanning peptide mutagenesis studies highlighted. Importantly, to date there is no substantive mutational data from human tumors to specifically support any of the downstream kinases, including the two AMPK catalytic genes, as being a particularly critical target of LKB1 in tumor suppression. One confounding issue with the lack of mutations found in these downstream kinases is that there is a great deal of redundancy among them, suggesting that loss of any one of them may be compensated for by other family members, unlike the case for LKB1 for which no other specific kinase has been shown to compensate in vivo.

Figure 3. AMPK and PI3K signaling converge to antagonistically regulate a number of downstream effectors, including the mTORC1 complex

A number of inherited hamartoma and cancer predispotion syndromes all share in common hyperactivation of mTORC1 or HIF-1α. Tumor suppressors inactivated in human cancer shown in light blue, oncogenes hyperactivated in human cancer shown in gold. Conditions that lower intracellular ATP levels (low glycolytic rates from low glucose or inhibitors like 2-deoxyglucose [2DG] or oxidative phosphorylation inhibitors like metformin and related biguanides) will lead to activation of AMPK in an LKB1-dependent manner. AICAR is a precursor of ZMP, which acts as an AMP-mimetic and is thought to directly bind the AMP-binding pockets of the AMPKγ subunit. A769662 is the only known small molecule that directly binds AMPK inducing its activity, though it is not currently known where the compound binds on the AMPK heterotrimer.

Figure 4. Control of cell polarity by LKB1-dependent signaling

The Par complex, composed of an atypical PKC family member, the Par-3 scaffold, the cdc42-binding Par-6, and cdc42 phosphorylates a number of downstream polarity proteins, including LKB1, the MARK family, and Lethal giant larvae (LGL). LKB1 also requires a signal from E-cadherin to be recruited and competent to phosphorylate AMPK at the adherens junction. LKB1-dependent AMPK activation is known to modulate the phosphorylation state of myosin light chain (MLC) in Drosophila mutants, which may be through indirect regulation of the kinase (MLCK) and phosphatase (MYPT1) for MLC. LKB1-dependent MARK kinases in turn phosphorylate the Par-3 scaffold, hence leading to the mutual exclusion of the Par complex and the MARK kinases within the cell. MARKs also are well-established to phosphorylate MAPs including tau, MAP2, and MAP4, and have been reported to phosphorylate DLG and Dishevelled (DVL) proteins in some contexts.