A Kinase Anchoring Protein 9 Is a Novel Myosin VI Binding Partner That Links Myosin VI with the PKA Pathway in Myogenic Cells.

Justyna Karolczak,Magdalena Sobczak,Maria Jolanta Rędowicz,Krzysztof Skowronek

doi:10.1155/2015/816019

Abstract

Myosin VI (MVI) is a unique motor protein moving towards the minus end of actin filaments unlike other known myosins. Its important role has recently been postulated for striated muscle and myogenic cells. Since MVI functions through interactions of C-terminal globular tail (GT) domain with tissue specific partners, we performed a search for MVI partners in myoblasts and myotubes using affinity chromatography with GST-tagged MVI-GT domain as a bait. A kinase anchoring protein 9 (AKAP9), a regulator of PKA activity, was identified by means of mass spectrometry as a possible MVI interacting partner both in undifferentiated and differentiating myoblasts and in myotubes. Coimmunoprecipitation and proximity ligation assay confirmed that both proteins could interact. MVI and AKAP9 colocalized at Rab5 containing early endosomes. Similarly to MVI, the amount of AKAP9 decreased during myoblast differentiation. However, in MVI-depleted cells, both cAMP and PKA levels were increased and a change in the MVI motor-dependent AKAP9 distribution was observed. Moreover, we found that PKA phosphorylated MVI-GT domain, thus implying functional relevance of MVI-AKAP9 interaction. We postulate that this novel interaction linking MVI with the PKA pathway could be important for targeting AKAP9-PKA complex within cells and/or providing PKA to phosphorylate MVI tail domain.

Highlights

Myosin VI (MVI) is a unique unconventional actin-based motor that unlike other known myosins moves towards the minus end of actin filaments [1, 2]
Several tissue specific MVI binding partners have been already identified in mammals; among them are proteins engaged in the regulation of cytoskeleton dynamics, proteins associated with the Golgi apparatus and BioMed Research International the endoplasmic reticulum, and proteins involved in endocytosis and cell adhesion as well as proteins with enzymatic activities [2, 4]
We have previously shown that MVI plays important roles both in skeletal and cardiac muscle [13, 15] and in C2C12 myoblasts [14]

Summary

Introduction

Myosin VI (MVI) is a unique unconventional actin-based motor that unlike other known myosins moves towards the minus end (i.e., backwards) of actin filaments [1, 2]. MVI belongs to a large myosin superfamily and has a domain organization similar to other known myosins; that is, it contains a motor, neck, and tail domain [3]. Its ∼140 kDa heavy chain is composed of the N-terminal motor domain (with the actin and ATP binding sites), a neck, to which two calmodulin molecules are bound, and a tail domain [1, 2, 4]. MVI exists as a monomer or a dimer, and it is believed that several factors such as cargo binding, monomer availability, and/or phosphorylation within the tail domain determine. Two regions of the MVI cargo domain were found to be involved in partner recognition: a positively charged RRL region and a hydrophobic WWY region. Several tissue specific MVI binding partners have been already identified in mammals; among them are proteins engaged in the regulation of cytoskeleton dynamics, proteins associated with the Golgi apparatus and BioMed Research International the endoplasmic reticulum, and proteins involved in endocytosis and cell adhesion as well as proteins with enzymatic activities [2, 4]

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Conclusion