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Einstein Quart. J. Biol. and Med. (2001) 18:54-58
Centrosomes as Scaffolds: The Role of Pericentrin and Protein
Kinase A-anchoring Proteins
Nicole E. Rempel
Department of Microbiology and Immunology
Albert Einstein College of Medicine
Bronx, New York 10461
tein kinase A (PKA) makes it an interesting scaffold for
regulation of cell cycle events and a possible target for
cancer therapy.
The Centrosome
Abstract
The centrosome, consisting of the centrioles and pericentriolar material, is a dynamic structure that organizes several proteins involved in cell cycle regulation.
Pericentrin and protein kinase A-anchoring proteins
(AKAPs) are proteins that associate into complexes at
the centrosome. Both bind protein kinase-A (PKA),
bringing PKA to the centrosomal subcellular compartment where it serves a regulatory function by interacting with proteins such as Op18/stathmin, which is
involved in microtubule dynamics. Pericentrin also binds
the microtubule nucleating γ-tubulin ring complex (γTuRC). This review presents recent findings that pericentrin and AKAPs act as scaffold proteins that recruit regulatory molecules to the centrosome.
Introduction
E.B. Wilson (1928) attributes the earliest discovery of the
centrosome to Theodor Boveri who, in 1901, described it
as a central body containing “centroplasm” and the centriole. Wilson continues, “in its simplest form the central
body appears under the highest powers as a single granule of extreme minuteness…at the focus of astral rays”
(Wilson, 1928). Since 1928, the role of the centrosome
has continued to be refined. It is now referred to as the
microtubule organizing center (MTOC) and is recognized
for its role in spindle formation, transport of cytoplasmic
vesicles, and regulation of cell shape (Zimmerman et al.,
1999). With the advent of techniques such as immunogold electron microscopy and improved immunofluorescence techniques, it is now possible to identify structural
proteins of Boveri’s “centroplasm.” These techniques
have unveiled new possibilities for the centrosome as an
organizer of cell cycle associated phenomenon.
This review focuses on the role of the centrosome in the
cell cycle and how specific proteins such as pericentrin
and protein kinase A-anchoring proteins (AKAPs) may
facilitate centrosome activity by serving as structural and
regulatory scaffolds for those proteins that regulate cell
cycle events (Figure 1). Pericentrin’s putative role as a
scaffold protein comes from structural analysis as well as
from studies that elucidate its interaction with complexes and signaling molecules at the centrosome (Doxsey et
al., 1994; Merdes and Cleveland, 1997; Dictenberg et al.,
1998). Pericentrin’s association with γ-tubulin and pro-
The centrosome appears as bodies of electron-dense
material either diffused throughout the cytoplasm or
specifically localized around centrioles. The centrosome
serves as the cell’s MTOC, a dynamic structure that
assumes different forms and locations depending on the
cell cycle phase (Brinkley, 1985). During the G1 phase of
the cell cycle the centrosome, or MTOC, consists of a single pair of organelles called centrioles. Each centriole is
constructed from nine triplet microtubules and forms at
a right angle to its partner centriole. At the G1/S boundary, when DNA replication commences, the centrioles
begin to separate slightly. At this time, each centriole
will duplicate semiconservatively, and the new centriole
will elongate during the G2 phase, again orthogonal to
its partner centriole. Two centriole pairs are now contained within one centrosome. At the initiation of the
mitotic phase, the centriole pairs move to opposite sides
of the nuclear envelope and recruit half of the pericentriolar material (PCM) with them (Kellogg et al., 1994).
As the nuclear envelope breaks down at the end of
prophase, the microtubules originating from the centrosomes begin to polymerize and depolymerize in a
process called dynamic instability (Mitchison and
Kirschner, 1984a, 1984b). As the tubulin dimer pool
decreases during polymerization, the probability of
depolymerization increases. Once depolymerization is
initiated, the microtubule is completely taken apart,
increasing the dimer pool and leaving a free nucleation
site (Mitchison and Kirschner, 1984a). Rates of polymerization and depolymerization depend on the state of the
tubulin dimer (GTP versus GDP) at the plus end of the
microtubule (Mitchison and Kirschner, 1984b). Dynamic
instability continues until kinetochores attach to the
mitotic spindle and align chromosomes on the
metaphase plate. The kinetochore is a specialized protein complex that assembles on a discrete region of the
chromosome called the centromere. Following chromosome alignment, the cell proceeds into anaphase
(Merdes and Cleveland, 1997).
In 1977, Gould and Borisy showed that the intact centrosome, consisting of centrioles surrounded by PCM, could
be isolated from any cell cycle phase and could function
as an organelle in itself. They observed the attachment
of microtubule arrays to centrosomes in both dividing
and interphase cells. Two observations suggested that
the anchoring of microtubules occurred at the PCM and
Centrosomes as Scaffolds
not at the centriole: 1) After recovery from a colcemid
block, native microtubules attached to the PCM and not
to the centrioles and 2) When PCM was removed, the
centrioles alone did not produce microtubule asters
when incubated with purified tubulin. Numerous proteins have now been found to associate with the centrosome. These include kinases, phosphatases, the p53
tumor suppressor protein, and the γ-tubulin structural
protein (Brinkley and Goepfert, 1998).
Pericentrin – A Structural Scaffold
Using antisera from scleroderma patients, Tuffanelli et al.
(1983) identified a 220 kDa protein that by immunofluorescence localized to the centrosome. This protein,
named pericentrin, was found to form an integral part of
centrosomes and non-centriolar MTOCs (Doxsey et al.,
1994). The amino acid sequence of pericentrin predicts a
tripartite structure with a central alpha-helical domain
flanked by C- and N-terminal non-helical domains
(Doxsey et al., 1994). This structure is similar to that of
intermediate filaments, suggesting that pericentrin may
dimerize in a coiled-coil leaving the C- and N-termini to
interact with associated proteins (Albers and Fuchs,
1992). The flanking domains contain a number of protein
kinase recognition sequences (Doxsey et al., 1994). Using
intermediate filament dimerization and filament formation patterns of the nuclear lamins as a model (Aebi et
al., 1986), pericentrin can be envisioned to wrap multiple
times around the centrioles. Such a structure could provide docking for multiple proteins at the centrosome
(Doxsey et al., 1994). By enhanced immunofluorescence
and image deconvolution, pericentrin’s three-dimensional structure appears as a reticular lattice structure which
increases in complexity and size as the cell cycle progresses from G1 to mitosis (Dictenberg et al., 1998).
Structurally, pericentrin could efficiently tether multiple
proteins and form a complex that could serve as a scaffold. When pericentrin is disrupted by injection of antipericentrin antibodies in mouse oocytes, the microtubule organization is also disrupted (Doxsey et al.,
1994). Mictrotubules now form arrays with loose foci
near the cell cortex. These results indicate that pericentrin influences aster formation in immature centrosomes
and is perhaps one of the requirements for microtubule
nucleation in vivo.
Pericentrin and γ-Tubulin – A Microtubule Nucleating
Complex
γ-Tubulin is an essential component of the centrosome
and links the centrosome to microtubules (Stearns and
Kirschner, 1994). γ-Tubulin was localized to ring structures in the PCM of purified centrosomes by immunogold electron microscopy (Moritz et al., 1995). Zheng et
al. (1995) showed that this γ-tubulin ring complex
(γ–TuRC) could nucleate microtubules in vitro through
55
binding at microtubule minus ends. The orientation of
the microtubule with respect to the γ–TuRC was determined by analyzing differences in the growth rates of
microtubules at their plus- and minus-ends. Given that
the plus end of the microtubule grows approximately
three times faster than the minus end in the presence of
free tubulin, the rates of the two ends were compared
after addition of γ-TuRC by pulsing varying concentrations of rhodamine-labeled tubulin. From these results, a
seed-nucleation model was proposed where the γ-TuRC
stabilizes the minus end of the growing microtubule at
the centrosome (Zheng et al., 1995).
Using Xenopus eggs as a source of centrosome components, Dictenberg et al. (1998) employed several biochemical methods to demonstrate the interaction of
pericentrin with γ-tubulin in a complex. Both pericentrin
and γ-tubulin were detected in high density sucrose gradient fractions. However, in vitro-transcribed pericentrin
migrated in the low density fraction. Pericentrin and γtubulin also co-eluted by gel filtration, indicating that
they are either part of the same complex or of different
complexes with similar biochemical properties.
Immunoprecipitation assays using Xenopus and mammalian cellular extracts confirmed formation of complexes between γ-tubulin and pericentrin. Nucleated
microtubules associated with the lattice in a predicted
ratio of one pericentrin complex to two γ-tubulin com plexes. From these data, Dictenberg et al. (1998) postulated that pericentrin may be necessary for γ-tubulin
complexes to assemble at the centrosome.
Pericentrin associates with γ-tubulin in complexes at the
centrosome and also influences microtubule nucleation
patterns. This suggests that either the correct binding of
γ-TuRC to pericentrin regulates the ordering of the
microtubule arrays or that other proteins that also bind
pericentrin help regulate γ-TuRC nucleating activity.
AKAPs – Regulatory Scaffolds
Also, the recently described centrosomal AKAPs serve a
scaffolding role at the centrosome (Witczak et al., 1999;
Schmidt et al., 1999). AKAPs act as scaffolds to recruit
PKA to subcellular compartments. PKA, which is made
up of regulatory (R) and catalytic (C) subunits (R2C2 in the
holoenzyme), is regulated by cyclic AMP (Beebe and
Corbin, 1986). Like pericentrin, the centrosomal AKAPs
have coiled-coil domains that are characteristic of structural proteins (Witczak et al., 1999). In 1999, both
Witczak et al. and Schmidt et al. described novel AKAPs
that localize to the centrosome and tether PKA by binding to the regulatory subunit (RII). AKAP450 was identified by RII overlay screening of a Jurkat T lymphocyte
expression library using 32P labeled RII as a probe and
also by using antibodies to rabbit AKAP120 (Witczak et
al., 1999). AKAP350 was also identified using rabbit
AKAP120 antibody and is involved in both centrosomal
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Einstein Quarterly Journal of Biology and Medicine
Figure 1: The centrosome and associated scaffold proteins. Pericentrin and AKAPs serve as scaffolds that sequester PKA at the centrosome. PKA in turn
affects microtubule organization by regulating proteins like Op18, a microtubule destabilizer. AKAP, protein kinase A-anchoring protein; Op18,
Oncoprotein 18; P, phosphorylated; PKA, protein kinase A.
and noncentrosomal regulation of cytoskeletal systems
by anchoring PKA (Schmidt et al., 1999). During the cell
cycle, AKAP350 was localized to the centrosome.
It was also found at the cleavage furrow in anaphase
and telophase cells, provided a possible role for
AKAP350 in the contractile ring that forms during
telophase (Witczak et al., 1999). The localization of
AKAPs and their ability to recruit PKA to subcellular
compartments implies a scaffold-type regulation of PKA
during many phases of the cell cycle. Specific PKA signaling is postulated to result at least in part from its proximity to substrate, and prevented cross-talk with other
PKA-signaling pathways (Pawson and Scott, 1997).
PKA has been shown to affect microtubule organization
(Melander-Gradin et al., 1998; Lane and Kalderon, 1994).
Mutations in PKA caused mislocalization of RNAs by disrupting microtubules in Drosophila (Lane and Kalderon,
1994). PKA has also been associated with microtubule
dynamics by its interaction with Op18/stathmin. Op18
interacts preferentially with unpolymerized microtubule
subunits, increasing the catastrophe (depolymerization)
rate and regulating microtubule dynamics in response to
external signals (Belmont and Mitchison, 1996).
Melander-Gradin et al. (1998) investigated the role of
PKA in phosphorylation of Op18 using ectopic expression of the c-myc-tagged catalytic subunit of PKA and
Flag-tagged Op18. Op18 phosphoisomers were detected
with anti-Op18 antibodies using native PAGE
(PolyAcrilamide Gel Electrophoresis) to separate Op18
according to differences in charge caused by phosphorylation at each of four identified phosphorylation sites.
Two of these sites (Ser-16 and Ser-63) are phosphorylat-
ed by PKA. Phosphorylation of these two serines was
shown to downregulate Op18 microtubule destabilizing
activity. This was determined by measuring cellular
microtubule polymer content in Op18 and PKA co-transfected cells (Melander-Gradin et al., 1998). In summary,
AKAPs are present in regions of high microtubule nucleating activity. PKA is recruited to the AKAPs where it can
influence microtubule dynamics by regulating microtubule-destabilizing factors.
Linking Structural and Regulatory Scaffolds
Diviani et al. (2000) elucidated a link between pericentrin and PKA. They showed that pericentrin alone can
anchor PKA by binding RII and tethering it at the centrosome, therefore, giving pericentrin a role similar to
AKAPs. Pericentrin, RII, and the catalytic subunit of PKA
were all detected in pericentrin immunoprecipitates
(Diviani et al., 2000). These results indicate that, similar
to AKAPs, pericentrin acts to concentrate PKA at specific
subcellular compartments.
Pericentrin and Cancer
Genetic instability, resulting from improper regulation at
the centrosome, can lead to tumor formation. Theodor
Boveri first proposed a role for centrosomes in cancer in
1914 and suggested that cancer cells could arise due to
improper activity or replication of centrosomes. The
result would be a gain or loss of chromosomes during
mitosis (Salisbury et al., 1999; Brinkley and Goepfert,
1998). Centrosome abnormalities can affect the cell in
multiple ways. They can lead to improper centrosome
segregation and affect cell polarity. Vesicular trafficking
Centrosomes as Scaffolds
57
can also become disoriented when multiple MTOCs are
present within the cell. Aberrant protein scaffolding due
to centrosomal abnormalities can lead to improper or a
complete lack of centrosome localization of cell cycledependent kinases, such as PKA, Cdc2, and check-points
such as p53 (Salisbury et al., 1999).
Press Inc., New York. pp. 43-111.
Abnormal pericentrin levels have been observed in tumor
cells. Pihan et al. (1998) used immunoperoxidase staining
with an antipericentrin antibody in tissues and in cell
lines from breast, lung, prostate, colon, and brain cancers. Normal cells in interphase labeled a single dot with
anti-pericentrin antibodies. In mitotic cells, a pair of dots
were observed, one at each pole. Tumor tissue had a
much higher level of pericentrin expression, forming multiple foci that are sometimes interconnected by atypical
pericentrin filaments. This diffuse, patchy pericentrin
label often localized to structures that contained no centrioles. Yet, all pericentrin-labeled structures were able to
nucleate the growth of new microtubule asters regardless of the size, number, or shape of the pericentrin foci
(Pihan et al., 1998). Overexpression or mislocalization of
pericentrin could sequester regulatory proteins away
from functional centrosomes, and this situation could
lead to defects in the mitotic spindle and chromosome
segregation, which is a hallmark of tumor cells.
1:145-172.
Belmont, L.T. and Mitchison, M. (1996) Identification of a protein that
interacts with tubulin dimers and increases the catastrophe rate of microtubules. Cell 84:623-631.
Brinkley, B.R. (1985) Microtubule organizing centers. Annu. Rev. Cell Biol.
Brinkley, B.R. and Goepfert, T.M. (1998) Supernumerary centrosomes and
cancer: Boveri’s hypothesis resurrected. Cell Motil. Cytoskeleton 41:281-288.
Diviani, D., Langeberg, L.K., Doxsey, S.J., and Scott, J.D. (2000) Pericentrin
anchors protein kinase A at the centrosome through a newly identified
RII-binding domain. Curr. Biol. 10:417-420.
Dictenberg, J.B., Zimmerman, W., Sparks, C.A., Young, A., Vidair, C.,
Zheng, Y., Carrington, W., Fay, F.S., and Doxsey, J.S. (1998) Pericentrin and
gamma-tubulin form a protein complex and are organized into a novel
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Doxsey, S.J., Stein, P., Evans, L., Calarco, P.D., and Kirschner, M. (1994)
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Gould, R.R. and Borisy, G.G. (1977) The pericentriolar material in
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Conclusions
Due to improved deconvolution algorithms, and
immunofluorescence techniques, several interesting proteins have been discovered at the PCM. Of these, pericentrin and the AKAPs are implicated in scaffolding roles
at the centrosome (Witczak et al., 1999; Schmidt et al.,
1999; Doxsey et al., 1994; Merdes and Cleveland, 1997).
The nature of these proteins allows one to envision the
PCM as a complex scaffold designed to coordinate proteins for proper and efficient function. Pericentrin may
serve as a structural scaffold necessary for microtubule
nucleation in cooperation with γ-TuRCs. It may also act as
a link between structural elements like the microtubule
network and regulatory elements like PKA and downstream targets. Determining the function of the centrosomal scaffold proteins, pericentrin, and the AKAPs, as
well as the proteins and signaling molecules they bring
together, may have important implications for understanding tumorigenesis.
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Kellogg, D.R., Moritz, M., and Alberts, B.M. (1994) The centrosome and
cellular organization. Annu. Rev. Biochem. 63:639-674.
Lang, M.E. and Kalderon, D. (1994) RNA localization along the aneroposterior axis of the Drosophila oocyte requires PKA- mediated signal
transduction to direct normal microtubule organization. Genes Dev.
8:2986-2995.
Melander-Gradin, H., Larsson, N., Marklund, U., and Gullberg, M. (1998)
Regulation of
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Merdes, A. and Cleveland, D.W. (1997) Pathways of spindle pole formation: different mechanisms; conserved components. J. Cell Biol.
138:953-956.
Mitchison, T. and Kirschner, M. (1984a) Microtubule assembly nucleated
by isolated centrosomes. Nature 312:232-237.
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