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Ankyrin domain of myosin 16 influences motor function and decreases protein phosphatase catalytic activity

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Abstract

The unconventional myosin 16 (Myo16), which may have a role in regulation of cell cycle and cell proliferation, can be found in both the nucleus and the cytoplasm. It has a unique, eight ankyrin repeat containing pre-motor domain, the so-called ankyrin domain (My16Ank). Ankyrin repeats are present in several other proteins, e.g., in the regulatory subunit (MYPT1) of the myosin phosphatase holoenzyme, which binds to the protein phosphatase-1 catalytic subunit (PP1c). My16Ank shows sequence similarity to MYPT1. In this work, the interactions of recombinant and isolated My16Ank were examined in vitro. To test the effects of My16Ank on myosin motor function, we used skeletal muscle myosin or nonmuscle myosin 2B. The results showed that My16Ank bound to skeletal muscle myosin (K D ≈ 2.4 µM) and the actin-activated ATPase activity of heavy meromyosin (HMM) was increased in the presence of My16Ank, suggesting that the ankyrin domain can modulate myosin motor activity. My16Ank showed no direct interaction with either globular or filamentous actin. We found, using a surface plasmon resonance-based binding technique, that My16Ank bound to PP1cα (K D ≈ 540 nM) and also to PP1cδ (K D ≈ 600 nM) and decreased its phosphatase activity towards the phosphorylated myosin regulatory light chain. Our results suggest that one function of the ankyrin domain is probably to regulate the function of Myo16. It may influence the motor activity, and in complex with the PP1c isoforms, it can play an important role in the targeted dephosphorylation of certain, as yet unidentified, intracellular proteins.

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Abbreviations

GSH:

Glutathione

GST:

Glutathione S-transferase

HMM:

Heavy meromyosin

MLC20:

Myosin regulatory light chain

MLCK:

Myosin light chain kinase

My16Ank:

Myosin 16 ankyrin domain

Myo16:

Myosin 16

MyPhoNE:

Myosin phosphatase N-terminal element

MYPT1:

Myosin phosphatase regulatory subunit 1

sk:

Skeletal

skMyo2:

Full-length skeletal myosin

NM2B:

Nonmuscle myosin 2B

PP1c:

Protein phosphatase-1 catalytic subunit

TEV:

Tobacco etch virus

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Acknowledgments

This work was supported by grants from the Hungarian Scientific Research Fund (OTKA grants NN 107776 and K112794 to M.N. and K109249 to F.E.) and by the “TÁMOP 4.2.4. A/2-11-1-2012-0001 National Excellence Program” supported by the European Union and the State of Hungary, co-financed by the European Social Fund to Z.K. and B.B. The authors thank Attila Nagy for preparing and providing NMIIB HMM.

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The authors declare that they have no conflicts of interest.

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Correspondence to Miklós Nyitrai.

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Kengyel, A., Bécsi, B., Kónya, Z. et al. Ankyrin domain of myosin 16 influences motor function and decreases protein phosphatase catalytic activity. Eur Biophys J 44, 207–218 (2015). https://doi.org/10.1007/s00249-015-1015-z

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  • DOI: https://doi.org/10.1007/s00249-015-1015-z

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