The principal findings of this study are that for NDPK-A, the phosphorylation status of a single amino acid, S122, determines whether NDPK-A channels ATP to AMPK α1, thereby regulating AMPK α1 activity towards an important regulator of fatty acid metabolism, ACC1. Additionally, we demonstrate that the presence of a glutamic acid at position 124 is both necessary and sufficient for this interaction with AMPK α1. The absence of E124 in NDPK-B explains our recent demonstration that NDPK-A (but not NDPK-B) specifically interacts with AMPK α1 and that this interaction allows NDPK-A-generated ATP (from GTP plus ADP) to be preferentially used by AMPK α1 as a phosphate donor in downstream AMPK mechanisms (6
). Importantly, we use the term “preferentially” to describe our finding that this locally generated ATP does not interact with ATP in bulk medium, suggesting that the NDPK-A/AMPK α1 complex partitions local ATP in some manner. Crucially, within this region, we find that NDPK-A only channels substrate and regulates ACC1, provided that S122 is capable of being phosphorylated or exists as a phosphomimic (Fig. ). Thus, AMPK α1 can be regulated by another protein in the absence of AMP.
Parallel data using null tissue further confirm the specificity of the NDPK-A/AMPK α1 interaction when liver cytosols from wild-type, NDPK-null, AMPK α1-null, and AMPK α2-null mouse tissue were compared (1
). We demonstrate that in the absence of AMPK α1 (but not AMPK α2), NDPK is unable to coprecipitate AMPK-SAMS activity. Conversely, in the absence of NDPK-A, substrate channeling does not occur, and only basal AMPK α1 activity (autophosphorylation) is present. Since gross amounts of AMPK α1 proteins are unaffected by the loss of NDPK-A, the absence of substrate channeling is likely to be functionally significant.
Functional relevance was investigated in two ways. First, to isolate the specific regions/residues of NDPK-A targeted by AMPK, we used four peptides corresponding to exposed regions of NDPK-A and found that AMPK was able to phosphorylate NDPK-A in only two regions, NDPKpep3 and NDPKpep4 (Fig. and ). Overlay binding analysis of these peptides revealed that AMPK α1 binds only the NDPKpep3 peptide. Second, the functional relevance of this interacting region was confirmed using ACC1 and NDPK-A precipitations (Fig. , right panel) to show that incubation with a molar excess of NDPKpep3 could disrupt the complex.
As shown in Fig. , we observed that within NDPKpep3, S122 and S144 on NDPK-A are AMPK targets in vitro. This notion was extended in vivo by immunoprecipitating NDPK-A from a human-derived liver cell line (HepG2) transfected with wild-type and AMPK-targeted point-mutated variants of NDPK-A (Fig. ). These cells were stressed with two different stimuli (phenformin and oligomycin) to activate AMPK. We observed that for untreated samples, the loss of S122, S144, and S120 resulted in the expected reduction in phosphoserine band intensity should they be bona fide in vivo targets for the relevant kinases. Analysis of the phenformin/oligomycin-treated cells demonstrated an increase in NDPK-A phosphoserine band intensity under wild-type, mock-transfected, and S120A conditions, but crucially, there was no change in the S122A and S144A mutants. Thus, we propose that S122 and S144 in NDPK-A are AMPK targets in vivo. Although further work will have to establish the role of S144 phosphorylation, the phosphorylation status of S122 may be important for the function of AMPK towards the in vivo target ACC1. This notion is supported by the differential change in the phosphorylation status of two established AMPK substrates (ACC1 and HSL) by comparing wild-type and NDPK-A-null tissues. We observed that the loss of NDPK-A from liver cytosol results in a reduction (~45%) in phospho-ACC1 compared with the wild type (Fig. ). Conversely, we observed that an AMPK α1 knockout results in an almost total loss of ACC1 phosphorylation (Fig. , upper left panel). The combined data indicate that while AMPK α1 is the primary driver of ACC1 phosphorylation in liver cytosol, NDPK-A is also responsible for a significant portion of that phosphorylation (approximately 45% compared to that of the wild type). Interestingly, the phosphorylation status of HSL in skeletal muscle is predominantly AMPK α2 dependent (32
), and as such, the loss of NDPK-A might not be expected to have any major effect on phospho-HSL levels. Nevertheless, the loss of NDPK-A reduced phospho-HSL by ~15 to 20% (Fig. , lower panels). The significance of this unexpected finding is beyond the scope of the current investigation.
The charge on the amino acid at position 124 on NDPK-A is negative (but is positive in NDPK-B), and we find this to be a critical determinant of the interaction with AMPK α1 (Fig. ). The glutamic acid at position 124 is highly conserved among NDPK isoforms, apart from NDPK-B. Thus, further work will have to determine whether NDPK-C, NDPK-D, etc., can also channel substrate via AMPK α1. Point mutation of residue 124 reciprocally (i.e., NDPK-A E124K and NDPK-B K124E) resulted in an NDPK-A mutant that could no longer bind or channel substrate to AMPK α1 and, conversely, an NDPK-B “pseudo-A” mutant that could now bind and channel substrate to AMPK α1. The generation of reciprocal pseudomutants of NDPK-A and NDPK-B allows us to speculate that NDPK-B may be channeling substrate in vivo to unknown associated proteins (similar to our reported NDPK-A/AMPK α1 interaction) by virtue of the fact that the core channeling mechanism remains intact (manifest by the ability of NDPK-B to channel substrate) (Fig. ). Such latent binding partners could be crucial for our understanding of unexplained differences between NDPK-A and NDPK-B functions. Further work will have to establish the significance of the finding that S122 is also present in NDPK-B but that this form fails to bind AMPK α1.
Figure crystallizes our observations and presents a working model showing that AMPK α1-dependent phosphorylation at S122 on NDPK-A determines whether NDPK-A is able to channel ATP to AMPK α1 and that E124 is required before this isoform-specific protein-protein interaction can proceed.
FIG. 6. Model of interaction between AMPK α1 and NDPK-A. The phosphorylation status of residue S122 on NDPK-A determines whether NDPK-A channels ATP derived from GTP plus ADP to AMPK α1 as the substrate in the phosphorylation of downstream AMPK (more ...)
Historically, the previously reported actions of AMPK have been described after pharmacological activation by 5-amino-4-imidazolecarboxamide riboside (13
), an AMP-mimetic agent that activates both α1 and α2 catalytic isoforms. In order to address this limitation, AMPK α1 and α2 knockout mice were recently generated (30
). Mice lacking the α1 isoform present no gross defect in glucose homeostasis, whereas mice lacking the α2 isoform exhibit glucose intolerance and reduced insulin sensitivity (31
). Therefore, investigation into the α2 knockout has proceeded apace, resulting in the establishment of a role for AMPK α2 in glucose homeostasis. This, taken alongside the known nuclear preponderance and greater AMP dependence of the α2 isoform, suggests that AMPK α2 functions in both short-term ATP conservation and long-term energy homeostasis and gene regulation (26
). Adding to the emerging variance in function between AMPK catalytic subunit isoforms, our data reveal a specific dynamic for AMPK α1 as part of a novel complex with NDPK-A and, crucially, the critical controller of fatty acid fat, ACC1. We speculate that this complex can rationalize and respond to levels of GTP additively to ATP (and possibly other triphosphates, although this remains to be tested), altering AMPK α1 activity without changes in AMP and irrespective of bulk cytosol ATP. We also report a downstream in vivo consequence in the control of cellular fatty acid metabolism. This finding is consistent with our previous finding that phosphate can be transferred from GTP to ACC1 via substrate channeling through NDPK-A (6
). This report also establishes that the AMPK α1 isoform is functionally linked to NDPK-A in a native tissue and that this complex is able to channel substrate towards only one of the two isoforms of AMPK that are involved in energy conservation. We suggest that substrate channeling could maintain AMPK activity under conditions of local ATP depletion (6
). It is therefore not unreasonable to propose that the two catalytic isoforms of AMPK might differentially address both long- and short-term energy homeostasis by distinct mechanisms.
Mice carrying a homozygous germ line-null mutation in the NM23-M1 gene (equivalent murine gene product to NDPK-A) have an unexplained phenotype. NDPK-A-null mice are smaller (20 to 25% lower weight than wild-type controls), display a high rate of neonatal mortality, and manifest altered mammary gland function in surviving adult females. Our previous work showed that ACC, a key regulator of fat metabolism, was part of the NDPK-A/AMPK α1 complex and that the substrate-channeling phenomenon described here altered the phosphorylation of ACC bidirectionally (6
). Our present work demonstrates that this complex can be disrupted by peptides corresponding to a small part of the NDPK protein (Fig. ). Since fat metabolism is essential for weight control and milk formation, we hypothesize that the weight reduction in NDPK-A-null mice might be linked to the loss of the NDPK-A/AMPK α1 complex mediated by the aberrant control of ACC and thus fat metabolism. Furthermore, since NDPK function is related to membrane turnover (15
), fatty acid synthesis (10
), breast cancer metastasis (19
), and mammary development (4
), this might explain defective mammary gland function in pregnant females who fail to feed their pups. Thus, our findings have a wide biological applicability given that NDPK-A function is also critical for the metastatic potential of breast cancer cells.
In Fig. , we model our understanding of the interaction of AMPK α1 with NDPK-A. We have incorporated our observation that an initial AMPK phosphorylation of S122 primes the NDPK-A/AMPK α1 complex towards substrate channeling and thus ATP conservation. Our findings shed light on molecular interactions of two critical cellular energy regulators, which could contribute to our understanding of the mechanism by which AMPK protects against cellular stress by decreasing fatty acid synthesis and increasing fatty acid oxidation. We propose that this is accomplished via NDPK-regulated AMPK-mediated phosphorylation of ACC (6
), creating a novel “fat controller.” Additionally, we propose that AMPK, the cellular fuel gauge, acquires an alternative fuel source in GTP via NDPK-A when cells are under stress.