AMP-activated protein kinase (AMPK) is an energy sensor that regulates cellular functions in response to changes in energy availability. However, whether AMPK activity is spatially regulated, and the implications for cell function, have been unclear. We now report that AMPK associates with the Golgi, and that its activation by two specific pharmacological activators leads to Golgi fragmentation similar to that caused by the antibiotic Golgicide A, an inhibitor of Golgi-specific Brefeldin A resistance factor-1 (GBF1), a guanine nucleotide exchange factor that targets ADP-ribosylation factor 1 (ARF1). Golgi fragmentation in response to AMPK activators is lost in cells carrying gene knockouts of AMPK-α subunits. AMPK has been previously reported to phosphorylate GBF1 at residue Thr1337, and its activation causes phosphorylation at that residue. Importantly, Golgi disassembly upon AMPK activation is blocked in cells expressing a non-phosphorylatable GBF1-T1337A mutant generated by gene editing. Furthermore, the trafficking of a plasma membrane-targeted protein through the Golgi complex is delayed by AMPK activation. Our findings provide a mechanism to link AMPK activation during cellular energy stress to downregulation of protein trafficking involving the Golgi.

LKB1 was discovered as a tumour suppressor mutated in Peutz-Jeghers syndrome, and is a gene involved in cell polarity as well as an upstream protein kinase for members of the AMP-activated protein kinase family. We report that mammals express two splice variants caused by alternate usage of 3'-exons. LKB1(L) is the previously described form, while LKB1(S) is a novel form in which the last 63 residues are replaced by a unique 39-residue sequence lacking known phosphorylation (Ser(431)) and farnesylation (Cys(433)) sites. Both isoforms are widely expressed in rodent and human tissues, although LKB1(S) is particularly abundant in haploid spermatids in the testis. Male mice in which expression of Lkb1(S) is knocked out are sterile, with the number of mature spermatozoa in the epididymis being dramatically reduced, and those spermatozoa that are produced have heads with an abnormal morphology and are non-motile. These results identify a previously undetected variant of LKB1, and suggest that it has a crucial role in spermiogenesis and male fertility.

Mammalian AMP-activated protein kinase is a serine/threonine protein kinase that acts as a sensor of cellular energy status. AMP-activated protein kinase is a heterotrimer of three different subunits, i.e. alpha, beta and gamma, with alpha being the catalytic subunit and beta and gamma having regulatory roles. Although several studies have defined different domains in alpha and beta involved in the interaction with the other subunits of the complex, little is known about the regions of the gamma subunits involved in these interactions. To study this, we have made sequential deletions from the N termini of the gamma subunit isoforms and studied the interactions with alpha and beta subunits, both by two-hybrid analysis and by co-immunoprecipitation. Our results suggest that a conserved region of 20-25 amino acids in gamma 1, gamma 2, and gamma 3, immediately N-terminal to the Bateman domains, is required for the formation of a functional, active alpha beta gamma complex. This region is required for the interaction with the beta subunits. The interaction between the beta and gamma subunits does not require this region and occurs instead within the Bateman domains of the gamma subunit, although the beta-gamma interaction does appear to stabilize the beta-gamma interaction. In addition, sequential deletions from the C termini of the gamma subunits indicate that deletion of any of the CBS (cystathionine beta-synthase) motifs prevents the formation of a functional complex with the alpha and beta subunits.

The MAP kinase pathway inhibitor U0126 caused phosphorylation and activation of AMP-activated protein kinase (AMPK) and increased phosphorylation of its downstream target acetyl-CoA carboxylase, in HEK293 cells. This effect only occurred in cells expressing the upstream kinase, LKB1. Of two other widely used MAP kinase pathway inhibitors not closely related in structure to U0126, PD98059 also activated AMPK but PD184352 did not. U0126 and PD98059, but not PD184352, also increased the cellular ADP:ATP and AMP:ATP ratios, accounting for their ability to activate AMPK. These results suggest the need for caution in interpreting experiments conducted using U0126 and PD98059.