AMPK Regulates Mitotic Spindle Orientation through Phosphorylation of Myosin Regulatory Light Chain

Abstract

The proper orientation of the mitotic spindle is essential for mitosis; however, how these events unfold at the molecular level is not well understood. AMP-activated protein kinase (AMPK) regulates energy homeostasis in eukaryotes, and AMPK-null Drosophila mutants have spindle defects. We show that threonine¹⁷² phosphorylated AMPK localizes to the mitotic spindle poles and increases when cells enter mitosis. AMPK depletion causes a mitotic delay with misoriented spindles relative to the normal division plane and a reduced number and length of astral microtubules. AMPK-depleted cells contain mitotic actin bundles, which prevent astral microtubule-actin cortex attachments. Since myosin regulatory light chain (MRLC) is an AMPK downstream target and mediates actin function, we investigated whether AMPK signals through MRLC to control spindle orientation. Mitotic levels of serine¹⁹ phosphorylated MRLC (pMRLC^ser19) and spindle pole-associated pMRLC^ser19 are abolished when AMPK function is compromised, indicating that AMPK is essential for pMRLC^ser19 spindle pole activity. Phosphorylation of AMPK and MRLC in the mitotic spindle is dependent upon calcium/calmodulin-dependent protein kinase kinase (CamKK) activity in LKB1-deficient cells, suggesting that CamKK regulates this pathway when LKB1 function is compromised. Taken together, these data indicate that AMPK mediates spindle pole-associated pMRLC^ser19 to control spindle orientation via regulation of actin cortex-astral microtubule attachments.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.00418-12.

ACKNOWLEDGMENTS

This work was supported in part by an American Cancer Society Research Scholar grant (RSG-08-035-01-CSM) awarded to A.I.M., grant 1R01CA142858 awarded to A.I.M., a Lung Cancer PO1 grant (5PO1 CA116676), and the Georgia Cancer Coalition.

Images were acquired at the Winship Cell Imaging and Microscopy Shared Resource. We thank Josh Francis for his assistance with the jasplakinolide experiment and Jennifer Fairman for the artwork in Fig. 11.