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Vol. 62, Issue 6, 1261-1273, December 2002
Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, Scotland, UK (A.M., M.D.H., S.J.Y.); Department of Biomolecular Sciences, University of Manchester Institute of Science and Technology, Manchester, UK (J.W.); and University of Alabama at Birmingham, Comprehensive Cancer Center, Birmingham Alabama (G.B.B.).
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Introduction |
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Our understanding of the
enigmatic receptor for activated C-kinase 1 (RACK1) protein has
increased dramatically in recent years from its original identification
as an anchoring protein for protein kinase C (PKC) (Ron et al., 1994a
).
By virtue of its ability to coordinate the interaction of key signaling
molecules, RACK1 is becoming widely perceived as playing a central role
in critical biological responses, such as cell growth. RACK1 is a 36-kDa protein (SwissProt accession no. P25388) containing seven
internal Trp-Asp 40 (WD40) repeats
(Fig 1A), with a consensus X6-94-[GH-X23-41-WD]N4-8
(where N = number of WD repeats). It is homologous to
the G protein
subunit, having 42% identity with many conserved
amino acid substitutions. The WD repeats of RACK1 can be
predicted to form a seven-bladed propeller structure (Sondek and
Siderovski, 2001
; Steele et al., 2001
), with each blade made up of
-sheets as shown in crystallographic studies for
G
(Wall et al., 1995
; Sondek et al., 1996
).
The WD repeat sequence of RACK1 is highly conserved in a diverse range
of species, including plants (Kwak et al., 1997
) and genetically
malleable species such as Drosophila melanogaster and
Caenorhabditis elegans (Bini et al., 1997
). Positioning of RACK1 WD repeats is even maintained in the alga
Chlamydomonas reinhardtii (Schloss, 1990
),
which diverged from the forerunners of the plant and animal kingdoms
some 600 million to 1 billion years ago. This has prompted the
suggestion that the biological function of RACK1 was established before
this separation occurred (Neer et al., 1994
). Indeed, RACK1 is
ubiquitously expressed in the tissues of higher mammals and humans
(Guillemot et al., 1989
), including brain, liver, and spleen,
suggesting that it has an important functional role in most, if not
all, cells (Chou et al., 1999
).
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RACK1 was originally cloned from both a chicken liver cDNA library and
a human B-lymphoblastoid cell line (Guillemot et al., 1989
) and
referred to as C12.3 or H12.3, respectively. The name RACK1 was adopted
by the Mochly-Rosen group to describe its ability to bind activated
PKC. This was because the rat gene product passed experimental criteria
similar to those used to identify protein A-kinase anchoring proteins
(Edwards and Scott, 2000
). These criteria were originally established
by Ron et al. (1994)
and recently refined by Dorn and Mochly Rosen
(2002)
: 1) injection of cells with purified RACK should block
PKC-mediated cell processes. Similarly, 2) delivery of peptides into
cells should block the interaction between a particular PKC isozyme and
its RACK, and this should specifically impair a known cellular function
of that isozyme. 3) Injection of peptides that induce an interaction
between a particular PKC isozyme and its RACK should selectively
activate that isozyme, and 4) RACK should bind PKC in the presence of
PKC activators (Ron and Mochly-Rosen, 1994
; Dorn and Mochly-Rosen, 2002
).
The first report on the structure and genomic organization of a
mammalian RACK was carried out on the porcine RACK1
gene (Chou et al., 1999
), which has almost 100% identity at the
protein level with its vertebrate homologs. The RACK1 gene
promoter contains a number of transcription factor binding sites
including serum response element, AP1, SP1, NF1, and YY1 (Chou et al.,
1999
). Binding of serum response factor to the serum response element is known to be essential for the transcription of certain genes in
response to growth factors; accordingly, RACK1 expression was found to
be up-regulated after serum stimulation (Chou et al., 1999
). That the
activity of the RACK1 gene is controlled by growth-promoting extracellular stimuli suggests that RACK1 may have a generalized role
in the cellular adaptation processes that occur during cell division.
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Diversity of Protein Interactions with RACK1 |
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RACK1 was originally found to interact with active
"conventional" PKC isoforms, with PKC
II seemingly being the
preferred binding partner (Ron et al., 1995
; Csukai and Mochly-Rosen,
1999
; Stebbins and Mochly-Rosen, 2001
). Conventional PKCs (
,
I,
II, and
) are calcium- and diacylglycerol-dependent protein
kinases that are activated after the receptor-stimulated hydrolysis of plasma membrane phosphatidylinositol 4,5-bisphosphate, which yields both calcium and diacylglycerol elevation (Mellor and Parker, 1998
).
Conventional PKCs, such as PKC
II (Fig.
2A), have in common a regular
organization of conserved protein domains (C1-4), interspaced with
isoform-specific, variable regions (Banci et al., 2002
). C2 regulatory
regions are found in a diverse range of proteins in addition to PKC
(Fig. 3B) and were the first protein
domains identified capable of interacting with RACK1 in a calcium- and phosphatatidyl serine-dependent manner (Banci et al., 2002
). The PKC
family is also represented by calcium-independent, "novel" PKCs
(
,
,
,
, and µ) and the diacylglycerol- and
calcium-independent (atypical) PKCs (
and
). RACK1 has been
reported to interact with novel PKCs, e.g., PKC
(Besson et al.,
2002
). These observations strongly support the notion that RACK1 may
regulate cell processes other than those involving conventional PKCs.
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Each PKC isoform displays distinct tissue and subcellular distributions
(Mellor and Parker, 1998
). However, the tissue distribution of RACK1 is
not always the same as its favored PKC (Chou et al., 1999
). This raises
the possibility that RACK1 may be involved in cell processes that are
independent of PKC signal transduction (Chou et al., 1999
). Indeed, the
accumulated data from a number of laboratories show that RACK1
interacts with a range of different cellular proteins and, as a result,
may have diverse, even cell-type-specific, functions (Table
1).
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The protein liaisons that involve RACK1 seem to fall into two broad
categories: constitutive, as with the cyclic AMP-specific phosphodiesterase PDE4D5 (Yarwood et al., 1999
), and
stimulus-dependent, as with PKC. The full range of domains that allow
protein partners to interact with RACK1 have yet to be determined;
however, Src homology (SH2) domains (Chang et al., 1998
) and pleckstrin
homology (PH) domains (Rodriguez et al., 1999
; Koehler and Moran, 2001
) have been identified as possible candidates. PH domains are protein modules of 100 to 120 amino acids, best known for their ability to bind
phosphoinositides (Lemmon et al., 2002
). SH2 domains are also modular
protein motifs of about 100 amino acids that interact with
phosphotyrosine residues on target proteins (Pawson et al., 2001
). The
fact that different sorts of protein domain can interact with RACK1
suggests that RACK1 has multiple docking sites. In addition, the
individual blades of the RACK1
-propeller may be able to direct
association with specific protein classes (Fig. 1B). The observation
that PH domains and activated PKC can bind concomitantly to RACK1
indicates that RACK1 indeed has multiple, independent protein binding
sites (Rodriguez et al., 1999
).
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How Might Specific Proteins Interact with RACK1? |
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Before the full-spectrum of protein liaisons that involve RACK1 can be determined, it will necessary to examine how specific protein domains direct interaction with RACK1. To date, little has been done in this area; however, pioneering work on PKC-RACK1 interaction from the Mochly-Rosen laboratory and recent investigations into the binding of PDE4D5 to RACK1 have begun to give us a glimpse of how RACK1 may coordinate some of its interacting partners.
Interaction of PKC with RACK1 is thought not only to target PKC to
appropriate intracellular locations but also to hold PKC in an active
conformation (Dorn and Mochly-Rosen, 2002
). This model is based on the
premise that the PKC isoforms that are capable of interacting with
RACK1 contain, within their primary amino acid sequence, a
"pseudoRACK1" binding site (Ron et al., 1994b
). It has been
proposed that the pseudoRACK1 site directs auto-regulatory interactions
allowing the pseudosubstrate site of PKC to interact with the
substrate-binding site, thereby helping to maintain the enzyme in an
inactive conformation (Ron et al., 1994b
). Peptides have been
discovered that can disrupt these interactions, thereby stabilizing the
bound PKC in an open, active conformation (Ron et al., 1994b
; Dorn and
Mochly-Rosen, 2002
). These peptides were derived from amino acid
sequences within the C2 domain of PKC, which is thought to contain at
least part of the RACK1 binding site, and from PKC-binding proteins
such as annexin (Ron and Mochly-Rosen, 1995
; Banci et al., 2002
). One
such example is the RACK1-derived peptide sequence DIINALCF, which is
derived from amino acids 234 to 241, in WD 6, of RACK1. Not only can
this peptide compete for the binding of PKC to RACK1 but also it can
activate the enzyme in vitro and in vivo (Ron et al., 1994b
; Ron and
Mochly-Rosen, 1994
). Peptides such as SIKIWD, which is derived from
amino acids 255 to 260, WD 6, of RACK1, only represent a
fraction of the PKC-binding site on RACK1 and are unable to stabilize
PKC in an active conformation (Ron et al., 1994b
; Dorn and
Mochly-Rosen, 2002
). The peptide SVEIWD, derived from amino acids 241 to 246 of the regulatory C2 region of PKC
, is a selective agonist of
PKC
function and the corresponding structural region within the
C2-domain of PKC
is thought to contribute significantly to the
formation of the auto-regulatory region of that enzyme (Ron and
Mochly-Rosen, 1995
; Banci et al., 2002
). Recent findings suggest that
the binding sites for RACK1 on PKC
II are found not only in the C2
domain of the protein but also in the V5 region of the enzyme (Fig. 2A) (Stebbins and Mochly-Rosen, 2001
). Accordingly, C2- and V5-containing peptide fragments also seem to inhibit the binding of RACK1 and PKC
(Ron et al., 1995
; Stebbins and Mochly-Rosen, 2001
). This suggests that
the molecular interactions that occur between PKC and RACK1 are complex
and may involve many points of contact between the two protein surfaces.
To try and put this peptide data in a structural context, we have
constructed a comparative model of RACK1 using bovine transducin G
as the structural template (Fig. 1A). From
this, we have identified an internal region that deviates markedly from
the G
structure (Lambright et al., 1996
). This
region occurs in blade 6 of the RACK1 propeller and has a significantly
longer inter-
-strand loop than the comparable region in
G
and may therefore have a role in determining
the binding specificity of RACK1 (Fig. 2B). The
-strand preceding
the loop region contains the SIKIWD sequence, which is thought to be
part of the PKC binding site on RACK1 (Ron and Mochly-Rosen, 1995
). If
this region in RACK1 were to contribute to binding PKC, then the outer
-strand of blade 6 would be required to adopt a nonblade
conformation. Our model suggests that the lengthened loop could enable
a degree of "conformational variability" in this region, with the
outer strand of this propeller blade swapping in and out of blade
conformation dependent on the presence of alternate binding partners.
Peptides from the V5 region of PKC have also been shown to contribute
to RACK1 binding (Fig. 2A) (Stebbins and Mochly-Rosen, 2001
). One of
these, QEVIRN (amino acids 645-650 of PKC
II), shares amino acid
homology with the outer
-strand of RACK1 blade 6, QEVIST (Fig. 2C);
however, 3D structure is not currently available for this region of
PKC. Our model presents the possibility that this region of PKC V5
could compete for and swap into the outer strand location on RACK1
blade 6. This model would also be consistent with an interaction
between the C2 and V5 domains of PKC (Keranen and Newton, 1997
;
Stebbins and Mochly-Rosen, 2001
; Banci et al., 2002
). In molecular
terms, this suggests that all these interactions could be mediated by
exchange of
-strands within the
-sheet framework of blade 6.
Looking at the loop that separates the
-strands in blade 6 of RACK1,
we see that the comparable loop in the PKC C2 domain mediates calcium
binding and contributes three ligands provided by Asp-246, Asp-248, and
Asp-254 (Fig. 2B) (Banci et al., 2002
). Interestingly, these ligands
align to acidic residues in the equivalent RACK1 region, when overall
alignment is determined by SIKIWD mapping to SVEIWD. In addition, the
glutamic acid in the QEVIRN sequence of the PKC V5 region maps to
Asp-254 of PKC C2 (Fig. 2B). This raises the possibility that calcium
ion binding may be involved in our suggestion of
-strand exchange
for interactions within PKC (between C2 and V5), as well as between
RACK1 and PKC. If such calcium involvement exists, it could, in
principle, add an additional regulatory element or simply represent
common interactions in each of the possible interacting combinations.
DIINALCF, one of the RACK1 sequences that has similarity to PKC-binding
sequences, partly forms the inner
-strand in the RACK1 blade 6 model
(Fig. 2B) (Ron and Mochly-Rosen, 1994
). Our model for outer-strand,
exchange-mediated interactions does not suggest a direct interaction
between the inner strand and PKC. However, in the framework of strand
exchange, it is possible that strand-forming potential alone could
supply a measure of binding affinity, and we suggest that this could
form the basis for some of the peptide binding data. All elements of
this model, from RACK1 structure to binding partner conformations and
calcium involvement, require testing with detailed biochemical and
structural analysis.
Yeast two-hybrid screens have been used to identify a number of novel
RACK1-interacting partners. An example of this is the cyclic
AMP-specific phosphodiesterase PDE4D5. This is one of a large family of
PDE isoforms (Houslay, 2001
). The PDE4 family is encoded by four genes,
each of which generates up to five isoforms that are distinguished by
unique N-terminal regions (Houslay, 2001
). The interaction between
RACK1 and PDE4D5 is extremely specific; PDE4D5 was not found to
interact with various other WD-repeat proteins and RACK1 does not
interact with any other PDE4 isoform (Yarwood et al., 1999
). We have
mapped the RACK1 interaction domain (RAID) in PDE4D5 to an 88-amino
acid N-terminal region that is unique to PDE4D5 (Yarwood et al., 1999
;
Bolger et al., 2002
). The predicted helical nature of the interaction
site raises the possibility that the binding of PDE4D5 to RACK1 may
occur in a manner analogous to the binding of G
to the WD repeat
protein G
(Fig. 4) (Steele et al.,
2001
). Mapping of the PDE4D5 interaction site on RACK1 by a combination
of yeast two-hybrid and N-terminal deletion analyses demonstrated that
WD repeats 5 to 7 of RACK1 are essential for it to interact with PDE4D5
(Steele et al., 2001
). However, a RACK1 construct generated from these
last three WD-repeats showed an interaction with PDE4D5 that was
approximately 25% as effective as wild-type RACK1 (Steele et al.,
2001
). This may indicate a minimum core unit for PDE4D5 interaction.
Whether this reduced interaction with PDE4D5 compared with RACK1 itself
represents poor folding or a requirement for additional sequence to
optimize interaction remains to be seen. In this regard, WD 1 is needed to complete the last blade of the propeller structure and loss of this
might underpin the poor efficacy of the N-terminal truncate (Fig. 1A).
Additionally, a reverse two-hybrid screen using these repeats
identified 11 single, nonproline amino acid mutations within this
region that nullify interaction with PDE4D5 (Steele et al., 2001
).
Mapping of these mutations onto our structural model of RACK1 indicates
that the amino acid residues essential for the RACK1/PDE4D5 interaction
predominantly cluster on the same face of RACK1 (Fig.
5) (Steele et al., 2001
). A large number of the mutations isolated in the screen were proline substitutions, which would be predicted to cause significant disruption of RACK1 folding. Indeed, our 3D-simulation of RAID bound to WD 5 to 7 of RACK1
demonstrates that these mutations occur at the blade interfaces for
intact RACK1, indicating that their effect may not be mediated directly
through RACK1-RAID interactions (Fig. 5). Because the mutations were
isolated in the WD 5 to 7 construct, rather than the full propeller
with its complement of blade interfaces, it is likely that stability of
the individual blade structures may be particularly sensitive to amino
acid changes. Intriguingly, many of the residues identified in RACK1 as
being important for interaction with PDE4D5 are conserved in the
primary structure of RACK1 from diverse species, including yeast (Fig.
6). However, the two phosphodiesterase
genes in yeast show no indication of being PDE4 homologs and have no
homology with the unique N-terminal region of PDE4D5 that directs
interaction with RACK1 (Wilson and Tatchell, 1988
; Matviw et al., 1993
;
Yarwood et al., 1999
). Given the remarkable degree of conservation of
these residues, it is possible that they are critical for the
structural integrity and proper function of RACK1, perhaps by
supporting the correct conformation of RAID binding sites.
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The requirement of these residues for RACK1 function requires further
clarification and is an important consideration when interpreting data
from experiments involving over-expression of RACK1. For example, in
the study by Buensuceso et al. (2001)
, alanine residues were introduced
into WD 6 of RACK1 in the putative PKC-binding site (DIINALCF) of RACK1
that, when over-expressed in CHO cells, reversed inhibition of cell
movement by wild-type RACK1. Although the authors claim that this
effect is through disruption of PKC binding to RACK1, one of the
residues converted to alanine was I236, a residue we now know to be
essential for interaction with PDE4D5 (Buensuceso et al., 2001
; Steele
et al., 2001
). Therefore, it could be argued that the effects observed in this study could also be caused by inhibition of PDE4D5 binding through overexpression of the mutant form of RACK1 or by disruption of
RACK1 structural integrity.
3D-modeling analysis of the interaction between
G
and G
has led
Sondek and Siderovski (2001)
to propose that various proteins binding
to the C-terminal region of
-propeller proteins may do so through a
G-
-like (GGL) motif. The cores of this are sequences DPLV and NPW
(Fig. 3). They suggest that the N terminus of PDE4D5, because of due to
the presence of similar motifs, might resemble a GGL domain and thus
bind to the C-terminal region of RACK1 in a manner similar to the
interaction between G
and G
(Fig. 3). We have found that alignment of
the amino acid sequences of PDE4D5 N terminus, the C2 domain of
PKC
II and the C2 domains of other proteins reveals a remarkable
degree of homology, particularly around the NPW motif of PDE4D5 (Fig.
3). Thus, by analogy with the GGL model, NPW may represent a common
core motif as seen with homologous proteins. If this is the case, then
the specificity of interaction must come from additional structural motifs that either enhance or reduce interaction with particular
-propeller proteins. The existence of this type of "structural conditioning" would mean that a particular family of
-propeller proteins could have different specificity with regard to their protein-binding partners. Additionally, for each
-propeller protein, there may be a family of proteins that can even interact at one `site'. From the 3D models presented here (Figs. 1, 2, 4, and 5), we
can see that WD 5 to 7 of RACK1 has a range of additional putative
interaction sites along a bifurcated groove and therefore a range of
possible modes of interaction, including those, like PKC, that interact
only with part of the surface, and those, like RAID, that interact with
multiple determinants over an extended surface.
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RACK1 Signal Transduction |
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PKC.
The in situ association and comovement of RACK1 and
PKC
II has been demonstrated in CHO cells treated with phorbol
12-myristate 13-acetate, in NG108-15 neuroblastoma cells (Ron et al.,
1999
), and in cardiomyocytes (Ron et al., 1995
). The localization of RACK1 was found to be very much cell-type dependent in both stimulated and unstimulated cells. That the localization of RACK1 alters concomitantly with PKC activation suggests that RACK1 does not anchor
PKC
II to one place but is probably involved in the shuttling of the
active enzyme to its appropriate subcellular site of action.
from the
cytosolic to particulate fraction after phorbol ester stimulation,
presumably because of the inability of PKC
to bind RACK1. The
inhibition of RACK1-mediated translocation of PKC is thought to
underlie the ability of DECA to delay morphological changes in
fibroblasts in response to phorbol ester treatment, to inhibit cell
motility and invasion, and to act as an antitumor agent. A potential
problem underlying such studies is that inhibitor peptides and small
molecules like DECA might be affecting the interactions between RACK1
and a number of different RACK1-binding partners because of homologies
in their RACK1-interaction domains.
PDE4D5.
The interaction of RACK1 with a critical regulator of
cAMP metabolism suggests that RACK1 may be intimately involved in the regulation of pathways activated by adenylyl cyclase. Indeed, the
adenylyl cyclase activator forskolin has been reported to cause RACK1
to localize to the nucleus, whereas PKC
II localization remains
unaffected (Ron et al., 2000
). RACK1 may therefore be involved in the
shuttling of non-PKC protein binding partners to the nucleus and may
play a role in cAMP-mediated gene expression. Another cAMP-elevating
agent, ethanol, which increases the activity of adenylyl cyclase,
thereby activating the cAMP/PKA signal transduction cascade (Saito et
al., 1985
), also induces the translocation of RACK1 to the nucleus (Ron
et al., 2000
). Ethanol also promotes the translocation of the catalytic
subunit of PKA to the nucleus (Dohrman et al., 1996
), and the
ethanol-induced compartmentalization of RACK1 is blocked by
adenosine-3',5'-cyclic monophosphorothioate, Rp-isomer, an
inhibitory analog of cAMP that prevents the activation of PKA. These
observations suggest that the recruitment of PDE4D5 to RACK1 may have a
pivotal role in regulating the activity of the fraction of cellular PKA
involved in regulating gene activity. Given the accumulation of
evidence from yeast and mammalian cell systems these genes will
possibly be those that are involved in the control of cell growth.
, as a permissive requirement for cAMP-regulation of CFTR channel
activity. The mechanisms underlying this phenomena are unclear because
the physiological target of activated PKC has yet to be defined
(Liedtke et al., 2002
together to enhance cAMP-control of CFTR.
Inactivation of PKC
, or displacement of PKC
from its binding site
on RACK1, would diminish cAMP-regulated CFTR function. It could also be imagined that recruitment of PDE4D5 to CFTR-associated RACK1 with the
possible displacement of PKC
would, by reducing local concentrations of cAMP, dramatically impair CFTR activity. Such a scheme may represent
a novel CFTR desensitization mechanism and a possible site for
therapeutic intervention
Tyrosine Kinases/Phosphatases.
In addition to interaction with
Ser/Thr kinases such as PKA and PKC, RACK1 also liaises with cellular
tyrosine kinases with possible growth-regulatory consequences. RACK1
was identified as a binding partner in a yeast two-hybrid screen to
identify proteins that interact with Src tyrosine kinase (Chang et al., 1998
). In vitro binding studies with glutathione
S-transferase (GST) fusion proteins revealed that two other
Src family tyrosine kinases, Lck and Fyn, also bind RACK1 and that
RACK1 binds to the SH2 domain of Src (Chang et al., 2001
). RACK1 and
Src were shown to coimmunoprecipitate from CHO cells transfected with
RACK1 and Src but not in cells cotransfected with RACK1 and a Src
mutant that has a three-amino acid deletion in the
phosphotyrosine-binding pocket of the SH2 domain, indicating that RACK1
interacts with the SH2 domain of Src in vivo. RACK1 and Src were also
demonstrated to coimmunoprecipitate from NIH3T3 cells using either
anti-RACK1 or anti-Src antibodies. The results of
coimmunoprecipitations using mutant RACK1, in which each tyrosine has
been individually substituted with phenylalanine together with
phosphopeptide competition assays, suggest that Src interacts with
phosphotyrosines in the sixth WD repeat of RACK1 (Chang et al., 2001
).
An in vitro protein kinase assay showed that GST-RACK1 could inhibit
Src activity in a concentration dependent manner, although it had no
effect on the activities of three Ser/Thr protein kinases (Chang et
al., 1998
). Levels of Src activity and tyrosine phosphorylation of many
proteins were markedly reduced in cells overexpressing RACK1. Fibroblasts stably overexpressing RACK1 were observed to grow more
slowly than wild-type cells. This lower growth rate in RACK1 overexpressing cells seems to have been caused by a prolongation of the
G0/G1 stage of the cell
cycle rather than an effect of necrosis or apoptosis. The authors
propose that RACK1 exerts its effect on the growth of NIH3T3 cells via
its inhibition of Src activity but acknowledge that this is only one of
the possible mechanisms by which RACK1 may influence cell growth (Chang
et al., 1998
).
in the
developing neurites and growth cones of retinal explants (Rosdahl et
al., 2002
1-integrin-associated kinase activity and association of Crk with
p130CAS but has no effect on IGF-1-activated IRS-1, Shc, phosphatidyl
inoitol-3-kinase, and extracellular signal-regulated kinase pathways
(Hermanto et al., 2002| |
RACK1 Cell Physiology |
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Cell Development. Homologs of RACK1 have been discovered in genetically malleable organisms such as D. melanogaster and yeast, providing an invaluable step toward elucidating its cellular functions. These investigations have begun to point toward a multifaceted role for RACK1 in cell physiological processes, which may be tailored to the requirements of individual cell types.
A fission yeast homolog of mammalian RACK1, cross-pathway-control (Cpc) 2, having 77% similarity with mammalian RACK1, was isolated in a yeast two-hybrid screen to identify proteins that interact with Pat1, a kinase that has no structural homolog in other organisms (McLeod et al., 2000
Cpc2
cells), while viable, display cell cycle abnormalities. These include
facets associated with mitotic delay, cell elongation, and defects in
conjugation and meiosis. Such cell cycle defects in
Cpc2 cells could
be rescued by expression of Cpc2 and also by expression of mammalian
RACK1, indicating that RACK1 and Cpc2 are indeed structural and
functional homologs (McLeod et al., 2000
Cpc2 cells, Pat1 kinase does not accumulate to high levels in the
nucleus as it does in wild-type cells and instead displays a prominent,
punctate cytoplasmic distribution (McLeod et al., 2000
, Cpc2 may regulate
Pat1 not by altering its catalytic activity but by influencing its
subcellular localization. Disruption of Pat1 targeting in
Cpc2 cells
may go some way to explaining why Cpc2 is not absolutely required for
yeast development but is essential for the timing and progression of
development. The phenotypes observed in cells lacking Cpc2 are similar
to a subset of phenotypes observed in cells with defects in the
stress-activated MAPK pathway and in cells expressing constitutively
activated Pat1 (McLeod et al., 2000Cell Movement and Growth.
The use of yeast cell models has
clearly demonstrated a link between Cpc2/RACK1 and cell cycle control.
In recent years, a number of studies have focused on the role of RACK1
in cell growth control mechanisms in mammalian cells. Overexpression of
RACK1 in NIH3T3 mouse fibroblasts has been found to cause a reduction in growth rate in both anchorage-dependent and -independent conditions because of a G1 delay, which correlates with
increased levels of the cyclin-dependent kinase inhibitors
p21Cip1/WAF1 and p27Kip1
(Chang et al., 1998
; Hermanto et al., 2002
). In addition, cells that
overexpress RACK1 demonstrate enhanced spreading, an increased number
of actin stress fibers, focal contacts, and enhanced tyrosine phosphorylation of both focal adhesion kinase and paxillin (Buensuceso et al., 2001
; Hermanto et al., 2002
). Conversely, reduction of RACK1
expression in NIH3T3 cells by antisense depletion blocked cell
spreading and inhibited growth factor-stimulated cell proliferation (Hermanto et al., 2002
).
-integrins identified WD repeats 5 to 7 of
RACK1, presenting further alluring evidence for an involvement of RACK1
in cell adhesion and movement (Liliental and Chang, 1998
-heterodimeric cell surface receptors that mediate binding of
cells to the extracellular matrix (ECM) (Skubitz, 2002
-integrins only upon phorbol
ester treatment, a stimulus known to enhance integrin-mediated cell
adhesion. The authors conclude that RACK1 may play a role in
membrane-cytoskeletal association by acting as a scaffold to recruit
other proteins to focal adhesion complexes (Liliental and Chang, 1998
, which has been shown to be important
for the control of integrin-dependent adhesion, spreading, and motility
of human glioma cells (Besson et al., 2002
to integrin
chains (Besson
et al., 2002
targeting to integrin
receptors, by antisense depletion of RACK1 or over-expression of a
truncated form of RACK that lacks part of the integrin binding region
(amino acids 204-317, containing WD repeats 6 and 7 and part of WD
repeat 5), leads to impaired adhesion and migration of cells (Liliental
and Chang, 1998
signaling is known to play an important role in angiogenesis and tumor
growth, it is suggested that the availability of RACK1 may be relevant
to the downstream signaling of PKC
in angiogenically active tissues
and may have a central role in tissue remodeling processes per se.
Immune Responsiveness.
Ligand-initiated activation of
superoxide anion generation by phagocytic cells such as neutrophils is
a key mechanism in the immune response, and it has recently been
proposed that PKC
II and RACK1 are involved in these critical
functions (Korchak and Kilpatrick, 2001
). Peptide inhibition of
RACKI/PKC
II complex formation and antisense depletion of RACK1 were
found to enhance superoxide anion generation in neutrophilic HL60
cells, suggesting that RACK1 may sequester PKC
II to negatively
regulate superoxide anion generation or may divert PKC
II to other
signal transduction pathways (Korchak and Kilpatrick, 2001
).
Intriguingly, almost all neutrophil inflammatory functions are
susceptible to inhibitors of PDE4 cyclic AMP phosphodiesterase
activity, the predominant PDE isoform in these cells (Zhu et al.,
1998
). Inhibition of PDE4 activity blocks oxygen radical release from
neutrophils stimulated with a range of ligands, including tumor
necrosis factor-
,
N-formyl-L-methionyl-L-leucyl-L-phenylalanine, and C5a, whereas the phagocytic respiratory burst is less susceptible (Souness et al., 2000
). Therefore, a complementary mechanism underlying some of the effects of RACK1-antisense treated HL60 cells may function
through the inappropriate intracellular targeting of PDE4D5, which is
also known to interact with RACK1.
-chain of the receptors for the hematopoietic
and inflammatory cytokines interleukin-3 (IL-3) and IL-5 and
granulocyte macrophage colony stimulating factor (GM-CSF). This
interaction was discovered in a yeast two-hybrid screen using the
-chain as bait and was verified by coimmunoprecipitation and
pull-down assays (Geijsen et al., 1999
with the receptor complex, it is not clear whether RACK1 mediates this interaction. RACK1 may therefore regulate other IL-3/IL-5/GM-CSF receptor signaling functions, for example activation of signal transducers and activators of transcription (Stats) 5a and 5b
(Mui et al., 1995
,
, and
mediate innate immune responses to viral
infection through IFN
R/IFN
R2 for IFN
and IFN
and
IFN
R/IFN
R2 for IFN
(Colonna et al., 2002
-subunit of the IFN
R (Colonna et al., 2002Brain Function.
Levels of RACK1 are a reduced by around 50%
in the brains of aged rats compared with adult or middle-aged rat
brains (Pascale et al., 1996
). This is accompanied by a loss of PKC
translocation, suggesting that a depletion of RACK1 contributes to the
functional impairment in PKC activity in aged rat brains. PKC isozymes
are expressed at high levels in the brain and are thought to be
important in memory and learning processes (Selcher et al., 2002
). In
particular, conventional PKCs are involved in the regulation of a
number of processes, such as neurotransmitter release, receptor
desensitization, ion channel flux, and synaptic efficiency, which are
known to undergo age-related modulation (Battaini et al., 1997
).
Intriguingly, the pathophysiology of Alzheimer's disease (AD) has been
reported to involve attenuated PKC activity and translocation (Masliah et al., 1991
; Matsushima et al., 1996
). RACK1 levels are decreased in
both soluble and membrane fractions from the brains of persons with AD,
whereas PKC
II levels are unchanged (Battaini et al., 1999
),
suggesting that the impaired PKC signal transduction pathway in brains
of persons with AD is related to a reduction in RACK1 protein levels.
Somewhat confusingly, an earlier article by Shimohama et al. (1998)
reported that RACK1 levels were not significantly affected in brains of
persons with AD, but this discrepancy may reflect a difference in the
brain areas examined
and
in the brains of morphine-treated rats (Escriba and
Garcia-Sevilla, 1999
and
has not been found for other proteins involved in
opioid signal transduction, such as G
,
G
, GRK2, adenylyl cyclase, and PKA (Nestler
and Tallman, 1988
II
from RACK1 and provoked movement of RACK1 to the nucleus, whereas the
compartmentalization of PKC
II remained unaffected (Ron et al.,
2000
II
localization is unchanged. Chronic exposure to ethanol is known to
result in neuroadaptive changes such as tolerance, craving, and
physical dependence, which are thought to be modulated to some extent
by the alteration by ethanol of the action of PKC on some of the major
neurotransmitters, including GABA, glutamate, glycine, and muscarinic
receptors (Weiner et al., 1994
II and RACK1 have been demonstrated to associate with
GABAA receptor
-subunits (Brandon et al.,
1999
II associated with
1/
3 subunits is
dramatically increased by phorbol ester treatment, suggesting that it
is activated PKC that is targeted to GABAA
receptors in neurons. PKC is known to phosphorylate
GABAA receptors and inhibit their function. That the PKC
II/
-subunit interaction is direct shows that it is
independent of RACK1; however, RACK1 may play an auxiliary role by
modulating the affinity of interaction between PKC
II and
GABAA receptors or an action of PKC other than
controlling receptor phosphorylation. In this respect, blocking
PKC/RACK1 interaction disrupts the modulation of
GABAA currents by 5-HT2,
suggesting that RACK1-mediated targeting of PKC to the vicinity of
GABAA receptors is required for serotonergic signaling (Feng et al., 2001| |
Conclusions and Future Perspectives |
|---|
|
|
|---|
The multiplicity of cell functions in which RACK1 has been implicated probably reflects the ability of this scaffold protein to interact with a wide range of signaling proteins. Analogous to Cpc2 in yeast cells, mammalian RACK1 isoforms seem to direct "cross-pathway-control" by integrating communication from different signaling pathways through the orchestration of protein-protein interactions.
The wide range of vital cell processes that involve RACK1 suggests that
studies into the function of this scaffold protein will continue to
form the basis of an exciting and burgeoning research field. The
question still remains, however, as to the specific combinations of
RACK1-interacting signaling proteins that control individual cell
functions. Many of the signaling proteins that bind to RACK1 target the
C terminus of the protein (Fig. 1B). This suggests that in intact
cells, there may be a degree of competition between signaling proteins
for interaction with RACK1. This implicates RACK1 as contributing to
the regulation of the balance of activation between conspiring or
antagonistic signaling pathways. Alternatively, individual proteins may
bind RACK1 at discrete intracellular sites. Implicit to this is the suggestion that it is the intracellular targeting of interacting partners, rather than RACK1, that is central to this. Thus PDE4D5, which is a predominantly cytosolic enzyme, will compete for binding to
a different pool of RACK1 than activated PKC, which is targeted to
particulate structures in cells (Yarwood et al., 1999
). Fine structural
detailing of the interaction interfaces between RACK1 and its various
binding partners will lead to the development of highly specific
protein complex disruption compounds that will facilitate the
identification of cell functions linked to particular intracellular
pools of RACK1. Because of their specificity, these compounds are
predicted to possess potent therapeutic potential.
| |
Footnotes |
|---|
Received July 22, 2002; Accepted September 19, 2002
Address correspondence to: Dr. Miles D. Houslay, Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, Wolfson Building, University of Glasgow, Glasgow, Scotland G12 8QQ, UK. E-mail: m.houslay{at}bio.gla.ac.uk
| |
Abbreviations |
|---|
RACK1, receptor for activated C-kinase 1;
PKC, protein kinase C;
PH, pleckstrin homology;
3D, three-dimensional;
PDE, phosphodiesterase;
RAID, RACK1 interaction domain;
GGL, G-
-like;
CHO, Chinese hamster ovary;
DECA, 1,1'-decamethylenebis-4-aminoquinaldinium diiodide (dequalinium);
CFTR, cystic fibrosis transmembrane regulator;
PKA, protein kinase A;
NMDA, N-methyl-D-aspartate receptor;
IGF, insulin-like growth factor;
Cpc, cross-pathway-control;
MAPK, mitogen-activated protein kinase;
ECM, extracellular matrix;
IL, interleukin;
GM-CSF, granulocyte macrophage-colony stimulating factor;
Stat, signal transducers and activators of transcription;
IFN, interferon;
AD, Alzheimer's disease;
GST, glutathione
S-transferase.
| |
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