Myosin VI and its interacting protein LMTK2 regulate tubule formation and transport to the endocytic recycling compartment
Margarita V. Chibalina
2
Matthew N. J. Seaman
2
Christopher C. Miller
1
John Kendrick-Jones
0
Folma Buss
2
0
MRC Laboratory of Molecular Biology
, Hills Road,
Cambridge
, CB2 2QH,
UK
1
Departments of Neuroscience and Neurology, The Institute of Psychiatry, Kings College London
,
London
, SE5 8AF,
UK
2
Cambridge Institute for Medical Research, University of Cambridge
, Wellcome Trust/MRC Building, Hills Road,
Cambridge
, CB2 2XY,
UK
-
Summary
Myosin VI is an actin-based retrograde motor protein that
plays a crucial role in both endocytic and secretory
membrane trafficking pathways. Myosin VIs targeting to
and function in these intracellular pathways is mediated by
a number of specific binding partners. In this paper we
have identified a new myosin-VI-binding partner, lemur
ce tyrosine kinase 2 (LMTK2), which is the first
n transmembrane protein and kinase that directly binds to
e
ic myosin VI. LMTK2 binds to the WWY site in the
CS terminal myosin VI tail, the same site as the endocytic
lle adaptor protein Dab2. When either myosin VI or LMTK2
C
f
o
l Introduction
a
n Endocytosis is essential for the uptake of vesicles containing
ru receptor-bound nutrients and signalling receptors from the
Jo plasma membrane. After internalisation these vesicles are
transported from the plasma membrane into the cell and fuse
with the early endosome where cargo, such as signalling
receptors, are sorted into multivesicular endosomes for
lysosomal degradation. Membrane pumps, channels and
nutrient receptors, such as the transferrin receptor (TfR), are
sorted into a recycling pathway back to the plasma membrane,
either via a rapid loop from an early endosome or via a longer
indirect route through the perinuclear endocytic recycling
compartment (ERC) (Maxfield and McGraw, 2004). Although
the exact molecular mechanisms involved in the delivery of
receptors to the recycling compartment and the transport back
to the cell surface remain to be elucidated, the small GTPase
Rab11 and the Eps15 homology domain (EHD) family of
proteins have been implicated in controlling this membrane
trafficking pathway (Ullrich et al., 1996; Sonnichsen et al.,
2000; Naslavsky and Caplan, 2005; Naslavsky et al., 2006).
The actin-based motor protein myosin Vb has also been
reported to associate with Rab11 and to regulate transport of
receptors out of the perinuclear ERC back to the cell surface
(Hales et al., 2002; Lapierre et al., 2001; Roland et al., 2007).
We show here that another class of myosins is required for
the delivery of cargo from the early endosome into the ERC.
Class VI myosins play crucial role(s) in membrane trafficking
pathways, because they are the only class that so far has been
shown to move backwards towards the minus end of actin
filaments in the opposite direction to all other myosins so far
is depleted by siRNAs, the transferrin receptor (TfR) is
trapped in swollen endosomes and tubule formation in the
endocytic recycling pathway is dramatically reduced,
showing that both proteins are required for the transport
of cargo, such as the TfR, from early endosomes to the
endocytic recycling compartment.
Supplementary material available online at
http://jcs.biologists.org/cgi/content/full/120/24/4278/DC1
characterised (Wells et al., 1999). Myosin VI is associated with
secretory and endocytic membrane compartments (Buss et al.,
2001; Buss et al., 1998; Warner et al., 2003; Aschenbrenner et
al., 2003) and several binding partners, responsible for
differential intracellular targeting and/or recruitment of myosin
VI, have now been identified. In the early endocytic pathway,
recruitment of myosin VI to clathrin-coated structures at the
plasma membrane requires Dab2 (disabled-2) (Morris et al.,
2002; Spudich et al., 2007) and to uncoated endocytic vesicles
requires GIPC (GAIP-interacting protein C-terminus) (Bunn et
al., 1999; Naccache et al., 2006). Myosin VI function in
exocytic membrane trafficking pathways needs the Rab8
effector protein optineurin, which links myosin VI to the Golgi
complex (Sahlender et al., 2005). In polarised epithelial cells
myosin VI is required for the transport of newly synthesised
membrane proteins containing a tyrosine-sorting motif to the
basolateral domain. These membrane proteins are sorted on
route to the basolateral plasma membrane in the endocytic
recycling endosome (Ang et al., 2004), where myosin VI and
optineurin, together with Rab8 and TfR have been localised
(Au et al., 2007).
In this study we have identified lemur tyrosine kinase 2
(LMTK2, also known as cprk, KPI-2, BREK, Lmr2, AATYK2
and KIAA1079) as a myosin-VI-binding partner and have
investigated the role(s) of the LMTK2myosin-VI complex in
endocytic and exocytic membrane trafficking pathways.
LMTK2 is a member of the lemur kinase group and is a
Ser/Thr-specific protein kinase with two predicted
transmembrane domains at its N-terminus, followed by a
kinase domain and a very long C-terminal tail domain (Wang
and Brautigan, 2002; Kesavapany et (...truncated)