New Roles for the LKB1-NUAK Pathway in Controlling Myosin Phosphatase Complexes and Cell Adhesion

Anna Zagórska,* Maria Deak, David G. Campbell, Sourav Banerjee, Mariko Hirano, Shinichi Aizawa, Alan R. Prescott, Dario R. Alessi*

*To whom correspondence should be addressed. E-mail: azagorska{at}salk.edu (A.Z.); d.r.alessi{at}dundee.ac.uk (D.R.A.)

This PDF file includes:

• Materials and Methods
• Fig. S1. Overexpressed NUAK1 and NUAK2 interact with endogenous MYPT1- PP1β complex.
• Fig. S2. Identification of a small region in NUAK1 containing the GILK motif that binds to the MYPT1-PP1β complex.
• Fig. S3. Alignment of GILK motifs in vertebrate orthologues of NUAK1 and NUAK2 and C. elegans UNC-82.
• Fig. S4. Endogenous AMPKα1 and AMPKα2 do not immunoprecipitate the MYPT1-PP1β complex.
• Fig. S5. Further characterization of binding of NUAK1 to PP1β.
• Fig. S6. NUAK1 coimmunoprecipitating with MYPT1 is phosphorylated at its T loop.
• Fig. S7. Sequence alignment of NUAK1 phosphorylation sites on MYPT1.
• Fig. S8. Phosphorylation of the MYPT1-PP1β complex by NUAK1.
• Fig. S9. Effect of the NUAK1 inhibitor BX795 and NUAK1 shRNA on cell detachment.
• Fig. S10. Effect of different substrata on cell detachment.
• Fig. S11. Localization of F-actin and MLC2 during detachment of Lkb1^+/+ or Lkb1^–/– MEFs.
• Fig. S12. Localization of F-actin and MLC2 during detachment of Nuak1^+/+ or Nuak1^–/– MEFs.
• Fig. S13. Localization of F-actin and MLC2 during detachment of HEK293 cells treated with BX795.
• Fig. S14. Inhibition of LKB1-NUAK1 signaling increases number of focal adhesions, stress fibers, and cell area in MEFs.
• Fig. S15. Basal MLC2 phosphorylation in LKB1- or NUAK1-deficient cells.
• Fig. S16. Comparison of the phosphorylation of MYPT1 by ROCK, NUAK1, AMPK, BRSK1, or MARK2.
• Fig. S17. Analysis of the NUAK2 K503R mutant.
• Fig. S18. Analysis of the NUAK1 interaction with PP1-targeting subunits.
• Fig. S19. The effect of the NUAK1 inhibitor BX795 and the ROCK inhibitor Y-27632 on phosphorylation of MYPT1.
• Fig. S20. Cell dissociation does not affect NUAK1 activity.
• Table S1. Proteins identified by mass spectrometry in NUAK1 immunoprecipitates.
• References

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