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Biochemical Society Transactions (2006) Volume 34, part 5
Control of growth factor receptor dynamics
by reversible ubiquitination
S. Urbé1 , J. McCullough, P. Row, I.A. Prior, R. Welchman and M.J. Clague
Physiological Laboratory, School of Biomedical Sciences, University of Liverpool, Liverpool L69 3BX, U.K.
Abstract
Activated tyrosine kinase receptors acquire ubiquitin tags. Ubiquitination governs receptor down-regulation
through interaction with components of the endosomal ESCRT (endosomal sorting complexes required for
transport) machinery that shepherds receptors into luminal vesicles of multivesicular bodies en route to
the lysosome. We have characterized two de-ubiquitinating enzymes that interact with components of this
machinery. AMSH [associated molecule with the SH3 domain (Src homology 3 domain) of STAM (signal transducing adapter molecule)] shows specificity for Lys63 - over Lys48 -linked ubiquitin and may act to rescue
receptors from taking the lysosomal pathway. In contrast, UBPY (ubiquitin-specific processing protease Y)
does not discriminate between Lys48 and Lys63 -linked chains and is required for lysosomal sorting.
Introduction
Upon activation, many receptor tyrosine kinases become
ubiquitinated following recruitment of the E3 ligase c-Cbl.
In the case of EGFR [EGF (epidermal growth factor) receptor], the resultant smeared band that is seen by Western
blotting was proposed to consist of a multiply mono-ubiquitinated form [1,2], although more recent MS analysis suggests a more complex polyubiquitin chain profile in which
Lys63 linkages predominate [3]. Note that these chains do not
target proteins for proteasomal degradation, in contrast with
the better characterized Lys48 -linked chains.
The first point of engagement of ubiquitinated receptors
with the endosomal MVB (multivesicular body) sorting machinery is with the Hrs (hepatocyte growth factor-regulated
tyrosine kinase substrate)–STAM (signal transducing adapter
molecule) complex [ESCRT (endosomal sorting complexes
required for transport)-0], of which both components contain
UIM (ubiquitin interaction motif) domains. This complex is
concentrated together with clathrin (with which Hrs interacts) in areas of the endosome covered with an unusual
flat coat-structure [4–6]. Two DUBs (de-ubiquitinating enzymes), AMSH [associated molecule with the SH3 domain
(Src homology 3 domain) of STAM] and UBPY (ubiquitinspecific processing protease Y), interact directly with STAM
through a common binding site within its SH3 domain [7,8].
We have characterized their activities and effects on the EGFR
down-regulation pathway.
Key words: associated molecule with the Src homology 3 domain of signal transducing adapter
molecule (AMSH), endocytosis, epidermal growth factor (EGF), signal transducing adapter
molecule (STAM), ubiquitin-specific processing protease Y (UBPY), ubiquitination.
Abbreviations used: CHMP, charged multivesicular body protein; DUB, de-ubiquitinating
enzyme; EGF, epidermal growth factor; EGFR, EGF receptor; ESCRT, endosomal sorting complexes
required for transport; Hrs, hepatocyte growth factor-regulated tyrosine kinase substrate; MVB,
multivesicular body; SH3 domain, Src homology 3 domain; siRNA, small interfering RNA; STAM,
signal transducing adapter molecule; AMSH, associated molecule with the SH3 domain of STAM;
UBPY, ubiquitin-specific processing protease Y; UIM, ubiquitin interaction motif; Vps, vacuolar
protein sorting.
1
To whom correspondence should be addressed (email [email protected]).
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Biochemical Society
AMSH
Enzymatic properties
AMSH belongs to the JAMM (JAB1/MPN/MOV34 metalloenzyme) family of DUBs, which are metalloproteases [9].
We used an in vitro reaction, combining purified protein
together with ubiquitin chains of defined linkage, to show that
AMSH has exquisite specificity for enzymatically produced,
wild-type Lys63 - over Lys48 -linked chains [10,11]. Moreover,
co-incubation with STAM markedly stimulated this activity
in a manner that requires an intact UIM domain [11] (Figure 1). This led to a model in which AMSH activity is coupled
with its interaction with STAM on endosomes, wherein
STAM may capture Lys63 -linked substrates through its UIM
domain and present them to AMSH.
AMSH interactions
In addition to STAM binding, we uncovered an interaction
with clathrin heavy chain through MS analysis of coimmunoprecipitating proteins. Using purified components,
we confirmed that this interaction was direct and targeted
against the terminal domain of clathrin. Furthermore, we
showed that STAM itself can also directly interact with
clathrin heavy chain [11]. A genome-wide yeast two-hybrid
screen suggested that the Drosophila homologue of AMSH
may interact with components of the ESCRT-III complex,
Vps24 (vacuolar protein sorting 24)/CHMP3 (charged MVB
protein 3) and Snf7/CHMP4 [12], that are involved in the final
stages of luminal vesicle formation. We confirmed the interaction between mammalian Vps24 and AMSH using purified
components and showed that it could be stabilized by coincubation with STAM [11]. Thus both AMSH and clathrin
are more deeply embedded in the MVB machinery proteininteraction network than hitherto appreciated (Figure 2).
The AMSH–STAM–Hrs complex can engage in a tripartite
Information Processing and Molecular Signalling
Figure 1 STAM stimulates the activity of the Lys63 -specific
ubiquitin isopeptidase, AMSH
AMSH (1 µM) was pre-incubated at 4◦ C for 30 min on its own, with
His-tagged STAM (2.5 µM) or with a His-tagged UIM point mutant
STAM-LSAA (2.5 µM), and then added to 250 ng of Lys63 (K63)-linked
tetra-ubiquitin (Ub4) for 15 min at 37◦ C.
Figure 3 MVBs in UBPY-depleted cells are attached to each other
via regularly spaced tethers that are not seen in control cells
(A) Arrows indicate unusual electron-dense tethers. (B) Higher magnification. Scale bars, 50 nm. Reproduced from [13] with the permission of
c 2006 American Society for Biochemistry and Molecular
the publisher. Biology.
Figure 2 AMSH interacts with multiple components of the
endocytic MVB sorting machinery
AMSH, STAM and Hrs all interact with clathrin heavy chain, which may
stabilize AMSH in the endosomal Hrs–clathrin coat. AMSH is also able
to act as a bridge linking ESCRT-0 with ESCRT-III by simultaneously
interacting with STAM and Vps24.
UBPY
Enzymatic properties
UBPY shows fairly equal activity against both Lys48 and Lys63 -linked ubiquitin chains [13,14]. The activity
against Lys63 chains is lost however when chains are made
using ubiquitin bearing point mutations at the other six
internal lysine residues [10].
UBPY knock-down
interaction with clathrin and AMSH may act as a bridge
between ESCRT-0 and -III.
AMSH knock-down
siRNA (small interfering RNA)-mediated knock-down of
AMSH enhances the rate of EGFR down-regulation in HeLa
cells [10]. Although it remains to be formally shown that this
is due to a loss of DUB activity, we have proposed a working
model whereby AMSH acts at an early stage of the sorting
process to reverse receptor ubiquitination prior to commitment to the lysosomal pathway, and thereby divert the
receptor to a recycling route. In the simplest sense, AMSH
can be thought of as merely opposing the E3-ligase activity
of c-Cbl.
In our hands, UBPY knock-down using either of two targeting siRNA oligonucleotides has multiple effects on the
endocytic pathway. Ubiquitin accumulates on endosomes
and is drained from the nucleus and cytosol [13,14].
EGFR and Met receptor down-regulation are both inhibited
as judged by immunofluorescence (EGFR) and Western-blot
analysis (EGFR and Met) [13]. Importantly, the effect on
EGFR degradation could be rescued by the expression of
siRNA-resistant UBPY [13]. This finding differs from that
of Mizuno et al. [14], who observed an enhanced rate of
degradation when UBPY knock-down was effected by a
stably transfected plasmid bearing a short hairpin-targeting
sequence. We noticed that UBPY knock-down results in partial loss of Hrs (50%) and, more importantly, severely
reduced (>90%) levels of STAM [13]. In the presence of a
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Biochemical Society Transactions (2006) Volume 34, part 5
proteasome inhibitor, we detect a smeared higher molecular
mass ubiquitinated form of STAM, the presence of which is
entirely contingent on UBPY knock-down. Thus one function of UBPY may be to stabilize STAM by editing
STAM-associated Lys48 -linked ubiquitin chains and thereby
protecting it from proteasome-mediated degradation. This
function cannot overlap with AMSH owing to the differing
specificities of the enzymes. The depletion of STAM may
partially account for the inhibitory effect on EGFR degradation; however, this phenotype cannot be rescued by mere
overexpression of STAM, suggesting that other targets remain
to be discovered.
A further striking feature of knock-down cells is that they
display marked differences in endosomal morphology, containing more multivesicular endosomal profiles with an
altered size distribution skewed towards a greater maximal
chord length. Electron microscopy also provides striking
views of large numbers of MVBs ‘stitched together’ along
extended areas of close contact by a regularly spaced repeating
unit of electron-dense material with a characteristic length
of 23.3 ± 3.1 nm (Figure 3) [13]. UBPY-depleted cells hence
present with a complex pleomorphic phenotype, suggesting
a key role for this DUB in endosomal dynamics.
that these two enzymes are both involved in the regulation
of growth factor receptor trafficking, while it is currently
unclear if they act in opposition to each other or rather in a
co-ordinated fashion at different stages of the pathway.
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AMSH and UBPY are two endosomal DUBs with differing
ubiquitin chain specificities. Depletion experiments suggest
Received 14 June 2006
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Biochemical Society