©2007 LANDES BIOSCIENCE. DO NOT DISTRIBUTE.
tion, transcriptional and post-transcriptional regulation, degradation rate and association
with specific inhibitor proteins, CKIs.
The cyclin-CDK complexes activity drives positively the progression of the cell cycle,
whereas, by binding to and inactivating cyclin-CDKs, CKIs exert a negative control
through the cell cycle.
Based on their sequence homology and specificity of action, CKIs are divided into two
distinct families: INK4 and Cip/Kip.2 Members of the INK4 family, namely p15Ink4b,
[Cell Cycle 6:9, 1053-1061, 1 May 2007]; ©2007 Landes Bioscience
Fulvio Della Ragione*
Department of Biochemistry and Biophysics “F. Cedrangolo”; Second University
of Naples; Naples, Italy
*Correspondence to: Fulvio Della Ragione; Department of Biochemistry and
Biophysics “F. Cedrangolo”; Second University of Naples; Via Costantinopoli, 16;
80138 Naples, Italy; Tel.: +39.081.5665812; Fax: +39.081.5665812; Email:
Original manuscript submitted: 03/13/07
Manuscript accepted: 03/13/07
Previously published online as a Cell Cycle E-publication:
p27Kip1, Cip/Kip family, cyclin-dependent
kinase, cyclin-dependent kinase inhibitor, cell
This work was supported in part by grants
from Associazione Italiana per la Ricerca
sul Cancro (AIRC), Progetti di Rilevante
Interesse Nazionale (PRIN), and FIRB.
A Fascinating Labyrinth
The progression through the phases of cell division cycle is regulated by different
cyclins and cyclin‑dependent kinases (CDKs) complexes. Due to their key function, the
activity of cyclin/CDK complexes is controlled by several mechanisms, including the inhi‑
bition by a number of proteins collectively defined CDK inhibitors or CKIs. Among the
CKIs, p27Kip1 represents a protein of central activity for the control of several phenotypes,
including proliferation, differentiation and malignant transformation.
p27Kip1 belongs to the growing family of “natively unfolded,” “intrinsically disordered”
or “intrinsically unstructured” proteins. The disorder proteins present a very large number
of possible conformations that, after the binding, converge to a well‑defined structure with
an extraordinary affinity for the target. As matter of fact, the absence of a pre‑existing
folding strongly facilitates p27Kip1 interaction with a number of targets.
Until recently, p27Kip1 has been solely viewed as a nuclear protein with the function
of modulating cyclin‑CDK activity and hence, cell cycle progression. However, exhaus‑
tive studies have now demonstrated that the protein plays additional roles outside of the
nucleus, including, particularly, the control of cell motility. Thus, the cellular localization
is of fundamental importance in p27Kip1 function. Accordingly, at least two different
mechanisms of degradation, occurring either in the nucleus or in the cytosol, have been
observed. Convincing evidences have demonstrated that p27Kip1 is a phosphoprotein
showing at least six to eight phosphorylatable residues. However, the precise functional
roles of the phosphorylations and the identification of the kinases responsible for the
post‑synthetic modifications are still debated. In this brief review, we will report the
Literature data that connect the post‑synthetic modifications of p27Kip1 with its function,
localization and metabolism. The picture that emerges demonstrates that several of the
pieces of the CKI metabolism are still nebulous.
The precise number, function and phenotype of cells present in a specific tissue are
under a strict control which results in an amazing balance between a resting status, prolif-
eration, differentiation (both terminal or reversible) and apoptosis.
The cell cycle of all post-embryonic eukaryotic cells is divided into four phases,
namely: G1 phase (prior to DNA synthesis), S phase (period of DNA synthesis), G2 phase
(between DNA synthesis and mitosis) and M phase (mitosis). Collectively, G1, S and G2
are called interphase, the cell cycle period distinct from division of the nucleus (mitosis)
and cytoplasm (cytokinesis). The length of S, G2 and M phases is remarkably similar in
many different cells, while the greatest variation is seen in the length of G1. At some point
late in G1, called restriction point (R point), a cell becomes committed to traverse the
remainder of the cell cycle. Variations in the cell cycle extension are mostly due to varia-
tions in the length of G1 phase up to the R point.
In eukaryotic cells, the transition between cell cycle phases is governed by a set of
cyclins and cyclin-dependent kinases (CDKs) complexes.1 The activity of cyclin-CDKs
complexes is controlled at multiple levels, including assembly of cyclin and CDK subunits,
inhibitory and activating phosphorylation and dephosphorylation events, cellular localiza-
homology and specificity of action, CKIs are divided into two
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©2007 LANDES BIOSCIENCE. DO NOT DISTRIBUTE.
preorganized R-helix by mutagenesis does not induce a significant
thermodynamic or kinetic disadvantage to the kinase inhibitory
capability.14 However, stabilization of the p27Kip1 preformed helix
by alanine mutagenesis down-regulates significantly the rate at which
the CKI inhibits cyclin A-CDK2.14 These findings underscore the
importance of quaternary interactions in the folding and binding
events of p27Kip1 and suggest that a biological advantage of the CKI
intrinsic disorder is the “speed” in the molecular identification events.
p27Kip1 Metabolism, An Intricate Web
p16Ink4a, p18Ink4c and p19Ink4d specifically inhibit the activity of
CDK4 and CDK6, whereas Cip/Kip members, that is p21Cip1,
p27Kip1 and p57Kip2, inhibit a broader spectrum of cyclin-CDK
Until recently, Cip/Kip family members were almost solely viewed
as nuclear proteins with a primary function of modulating cyclin-CDK
activity and hence, cell cycle progression. However, emerging studies
now clearly indicate that Cip/Kip proteins play additional roles
outside of the nucleus. Particularly, all the members of Cip/Kip family nucleus. Particularly, all the members of Cip/Kip family
seem to affect, although at different levels, cell motility.
This heterogeneity of effects is associated to an intricate puzzle
of post-synthetic modifications, which appear to control the cellular
localization of the inhibitors, their interaction with other proteins
and, finally, their physiological role.
Following, we will review some findings on the metabolism of
p27Kip1 in an attempt to describe a putative interplay between its
metabolism and function.
p27Kip1 PROtein AnD its FunCtiOns
The protein domains. The p27
located on chromosome 12p13 at the junction of 12p12-12p13.1.9
The gene was cloned by several groups in 1994.6,7,10 A detailed
examination of the protein identified two major regions, the
N-terminal which, on the basis of homology with p21Cip1 and
p57Kip2, showed the capability of inhibiting the kinase activity
of cyclin-CDK complexes and a C-terminal region with initially
The crystal structure of the 69-residues amino-terminal inhibitory
domain of p27Kip1 bound to the phosphorylated cyclin A-CDK2
showed that p27Kip1 binding causes large conformational changes
in and around the catalytic cleft of CDK211 and that p27Kip1 has
separate binding sites for the cyclin and the kinase. This explains how
p27Kip1 can interact with isolated subunits.11,12 Binding of p27Kip1
to cyclin-CDK complex is significantly tighter than binding to either
cyclin or kinase subunit, which is consistent with cooperative binding
of the two subunits.
The crystal structure of the human p27Kip1 CDK-inhibitory
domain (residues 22–106) bound to human cyclin A-CDK2 shows
that residues 25–93 of p27Kip1 bind in an ordered conformation
comprising two helices and elements of b-structure.11 However, the
p27Kip1 CDK-inhibitory domain lacks an intramolecular hydro-
phobic core in the context of the cyclin A-CDK2 complex,11 and
circular dichroism and fluorescence spectra show that the unbound
domain is largely unfolded in both ideal and thermodynamically
nonideal, crowded solutions.13 A recent study has described the
functional consequences of conformational preferences in the intrin-
sically disordered human p27Kip1 CDK-inhibitory domain. Rather
than existing as a random coil, the p27Kip1 CDK-inhibition domain
exhibits a propensity to form preexisting helical structure that corre-
sponds to the R-helix, but not to the 310 helix, that forms upon
binding to cyclin A-CDK2.14 Alteration in the unbound state of the
he p27Kip1 gene (formally CDKN1B) is
Interestingly, many proteins involved in pivotal cellular processes
exhibit characteristics of unfolded, rather than folded, proteins.15,16
These proteins have been defined “natively unfolded”, “intrinsically
disordered” or “intrinsically unstructured”.15-18 A number of them
undergoes folding transition from disorder to order upon binding
target proteins or nucleic acids,15 showing a functional requirement
for organized structure for at least certain intrinsically disordered
proteins. Molecular recognition by intrinsically disordered proteins is
therefore similar to the folding process in that a very large number of
possible conformations converge to a well-defined structure.
The C-terminal domain of p27Kip1 has been reported to interact
with several proteins involved in processes apparently not correlated
to the cell cycle control. In particular, when localized at cytosolic
cellular compartment, p27Kip1 interacts, probably by means of the
C-terminal domain, with various proteins including RhoA, Rac,
Stathmin, Grb2 and 14-3-3 (Fig 1).
Numerous independent pieces of information demonstrated that
p27Kip1 modulates the structure of cytoskeleton and cell contractility
and mobility. Following are some details on the CKI regions puta-
tively involved in these activities, while the functional meanings of
the interactions are reported elsewhere in the text.
(1) p27Kip1-null fibroblasts are largely refractory to reorganize
their actin cytoskeleton and focal adhesion fibers in response to
growth factors and show an increased Rho-dependent phenotype
(i.e., a significant decrease in motility associated with an increased
numbers of actin stress and focal adhesions fibers and an up-regu-
lation of phosphorylation of cofilin, a target of the Rho-dependent
pathway).19 Although the interaction of p27Kip1 C-terminus with
RhoA has not mapped precisely, the binding has been demonstrated
by coimmunoprecipitation experiments, suggesting that the CKI
might affect RhoA directly.19 In particular, detailed experiments
allow the conclusion that p27Kip1 inhibits the Rho pathway by
blocking the GEF-mediated activation of RhoA.19
(2) Hepatocyte growth factor (HGF) treatment of HepG2 cells
induces cell migration associated with increased cytosolic p27Kip1
levels.20 The CKI colocalized with F-actin and the forced up-regu-
lation of the CKI is sufficient for actin cytoskeletal rearrangement
and migration of HepG2 cells. Loss or disruption of the p27Kip1
C-terminal domain abolishes both actin rearrangement and cell
migration. Rac GTPase appears necessary for p27Kip1-dependent
movement, but alone was insufficient for HepG2 cell migration.20
The region of p27Kip1 required for the Rac-related function is the
so-called “scatter domain” identified between aa 118–158 (Fig. 1).20
(3) p27Kip1 has been described as an inhibitor of cell migration
in HT-1080 fibrosarcoma cells and normal mouse fibroblasts.21
Interestingly, this study demonstrates the interaction of p27Kip1 with
stathmin, which is a microtubule-destabilizing protein. Binding of
p27Kip1 to stathmin leads to the inhibition of stathmin function and,
as a consequence, to the accumulation of stabilized microtubules.21
The interaction between p27Kip1 and stathmin requires aa 170–198
of p27Kip1 (Fig. 1).
The interaction of Grb2 and p27Kip1 has been described for the
first time in 2001 as a result of a “two-yeast hybrid screen” study
devoted to identify proteins interacting with the C-terminal domain
of p27Kip1.22 The p27Kip1 region involved in this binding mostly
includes the proline rich domain (aa 90–96) at the C-terminus of the
CKI (Fig. 1).22,23 In doing so, p27Kip1 competes with the Ras-GEF
Sos for binding to Grb2, thereby preventing Ras activation.22 Ras
activates multiple signalling cascades and is a potent inducer of cell
motility.24 However, the physiological relevance of this p27Kip1
interaction remains to be investigated.
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©2007 LANDES BIOSCIENCE. DO NOT DISTRIBUTE.
The function of cell cycle regulation is consistent with the obser-
vation that p27Kip1 homozygous null mice are viable but are bigger
than wild-type littermates.34-36 Moreover, adult mice lacking p27Kip1
present multiple organ hyperplasia, including pituitary tumors and,
in some cases, retinal dysplasia, thymic hyperplasia, female sterility,
and hyperplasia of the adrenal gland.34-36 In conclusion, p27Kip1
does not seem a vital gene, but a protein that controls, through its
p27Kip1 Metabolism, An Intricate Web
The 14-3-3 proteins are a family of abundant, widely expressed
28–33 kDa acidic polypeptides. These proteins bind to phospho-
serine/threonine-containing motifs in a sequence-specific manner and
function as adaptors regulating the activity of components involved
in signal transduction and cell cycle. So far, the interaction of 14-3-3
proteins with more than seventy proteins has been described, but
likely this value represents only a fraction of the physiological 14-3-3
partners. In 2002, Fujita and colleagues25 demonstrated that p27
interacts with various members of 14-3-3 family, namely 14-3-3s,
q, e, h.25 Subsequently, the results were confirmed by additional
studies demonstrating that the AKT-dependent phosphorylation on
threonine 157 or 198 induces the interaction between p27Kip1 and
14-3-3 protein (Fig. 1).26,27 In turn, the formation of the complex
should inhibit the entry of the CKI into the nucleus, thus inducing
its cytosolic accumulation.
Finally, two additional region have been identified in the p27Kip1
sequence, namely (1) a bipartite nuclear localization signal (NLS,
152/153-166/168) (Fig. 2) which is recognized by the alpha/beta
importins, allowing p27Kip1 transport into the nucleus27,28 and (2)
the putative nuclear export signal (NES) identified between amino
acids 32 and 45 (Fig. 2). However, other residues of the protein
could be involved in the export process including serine 10 and
The role of p27Kip1 in the control of cell growth. p27Kip1 has
been first described as an inhibitor of the activity of cyclin E-CDK2
and cyclin D-CDK4 complexes in response to transforming growth
factor-b.6,7 By this mechanism, the CKI modulates the G1to S phase
transition, causing the inhibition of pRb phosphorylation.31-33
So far, the interaction of 14-3-3
local inhibitory action on the cell cycle,
both body and organ size.
The cell cycle kinetics of mouse embry-
onic fibroblasts lacking the CKI is similar
to wild-type counterparts, but their CDK2
activity is strongly elevated. Accordingly
with this finding, most if not all phenotypes
of p27Kip1-deficient mice were thought as
dependent on increased CDK2 activity.
However, CDK2 ablation in p27Kip1-de-
ficient mice does not rescue the phenotypes,
thus allowing the hypothesis that CDK1 is
a further in vivo target for the CKI.37
p27Kip1 control of cell cycle regulation
is particularly important because the CKI
functions as a tumor suppressor that is
often disrupted in human cancers, usually
by compromising its stability and/or loca-
tion.38-41 Consistent with this view, mice
without p27Kip1 develop pituitary tumors
and display increased susceptibility to
Surprisingly, mice lacking a single copy
of the p27 p27Kip1 gene are still hypersensitive
to carcinogens, pointing up to the impor-
tance of precisely controlling its levels and activity.42-44 Despite these
observations, the role of p27 p27Kip1 in tumorigenesis remains mysterious
and probably due to its multifunctionality.
The role of p27Kip1 in apoptosis and cell motility. The relation-
ship between p27Kip1 and apoptosis remains ambiguous. In a number and apoptosis remains ambiguous. In a number
of cancer cell lines, overexpression of p27Kip1 induces a pro-apoptotic
effect.45 Accordingly, in solid cancers treated with inhibitors of
proteasome activity, cells showing high p27Kip1 levels became apop-
totic, while the nonmalignant cells did not.46,47 However, p27Kip1
plays an anti-apoptotic role in carcinoma and leukemic cell lines
during drug-induced apoptosis as well as in normal fibroblasts and
mesangial cells (from p27Kip1 null mice) cultured without growth
factors.42 Therefore, the influence of p27Kip1 on apoptosis appears
to be dependent on the experimental models and the data reported
do not allow any general and definite conclusion. Moreover, whether
or not the influence of p27Kip1 on apoptosis is dependent on its
effects on cell cycle remains to be clarified. In this context, it has
been demonstrated that caspase 3 cleaves p27Kip1,48 and that its
cleavage is not associated with CDK2 activation or cytoplasmic
relocalization.48 Finally, it is noteworthy that the overexpression of
p27Kip1 cleavage products prevents apoptosis, possibly through the
inhibition of cytochrome c release.49 These findings suggest a cell
cycle independent function of p27Kip1 on apoptosis. Further investi-
gations will be needed for a better understanding of p27Kip1 role in
cell programmed death.
Although confirmed in several instances, the function of p27
in cell motility seems to be strongly dependent on cellular pheno-
types, in that, in some cell types, p27Kip1 increases cell motility,
whereas in others decreases it. For example, ectopic overexpression
of p27Kip1 stimulates the migration of hepatocellular carcinoma
cells,20,50 while it inhibits migration in other cell types, including
vascular smooth muscle cells, umbilical vein endothelial cells,
neurons, and oral cancer cells.51-55 These differences might derives
from cell type-specific context and, particularly, from the relative
balance between Rho and Rac activity.
Figure 1. Major interactors of p27Kip1 and their putative functions. In the scheme, the amino acid
residues (as numbers) of p27Kip1 and their cellular interactors are reported. The narrow means that a
single amino acid has been demonstrated to be directly involved in the binding. When a stretch of the
protein is implicated in the interaction, it is reported as a line. Moreover, the sequence of the proline‑rich
sequence is shown in detail. In the case of RhoA, the precise region of the interaction has not been
mapped (represented as question marks). The putative function of the binding is shown in the respective
box. Further details are in the text.
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vated by ROCK phosphorylation. Therefore, although they act
differently, all of the Cip/Kip proteins, when localized in the cytosol,
seem to control negatively the Rho signaling pathway.
It is tempting to hypothesize that the modulation of different
proteins in the Rho pathway by CKIs could furnish new levels of
control of Rho-mediated processes. As matter of fact, the abundance
and subcellular localization of CKIs are regulated throughout the
cell cycle. An additional factor of complexity is the observation that
p27Kip1 Metabolism, An Intricate Web
Cell migration involves the sequential disassembly and reas-
sembly of focal adhesions fibers, with focal adhesion fiber turnover
at the leading edge allowing for cell protrusion. After contraction
and translocation of the cell body, a similar localized turnover of
focal adhesions allows for retraction of the trailing edge. The Rho
family GTPases coordinate these events. Cdc42 stimulates filopodia
formation and directed cell movement, Rac stimulates lamellipodia
formation at the leading edge, and Rho stimulates stress fiber forma-
tion and contraction.
Efficient cell migration needs that the activities of Rho and Rac
be tightly balanced; the dynamic activation and inhibition of both
GTPases are spatially and temporally coordinated. Thus, insufficient
levels of Rho-GTP will inhibit migration by preventing cells from
achieving the level of adhesiveness and traction they need to
Conversely, excessive Rho activity can also inhibit cell migration
by increasing adhesion and preventing focal adhesion turnover.58-62
The influence of p27p27Kip1 on Rho appears at present more defi-
nitely demonstrated than that on Rac. Indeed, a direct interaction by
means of coimmunoprecipitation has been reported between the two
proteins.19 At present, it has been demonstrated that p27Kip1 reduces
Rho-GTP levels and in turn the activity of ROCK (a RhoA effector)
resulting in a decrease of stress fiber formation and focal adhesion
assembly.63-66 However, it is likely that a strong down-regulation of
p27Kip1 might result into an exaggerate Rho activity, thus also causing
an altered balance and inefficacious contractility and movement.
Interestingly, it appears that all of the members of the Cip/Kip
family may play a role in the regulation of the Rho pathway, albeit
acting at distinct levels in the pathway. p27Kip1 regulates Rho activa-
tion while cytoplasmic p21Cip1 directly inhibits ROCK, resulting
in increased neurite outgrowth.67,68 Finally, p57Kip2 binds to
LIM-kinase and induces its translocation to the nucleus, thereby
inhibiting its activity.69 LIM-kinase phosphorylates and inactivates
the actin depolymerization factor cofilin, and is itself directly acti-
RhoA down-regulates the levels of p27Kip1
and p21Cip1 proteins.70-73 Thus, Rho and
p21Cip1 and p27Kip1 seem to participate in
a regulatory feedback loop, in that while
CKIs negatively control the Rho pathway,
Rho signaling causes the down-regulation
of the CKIs.
The network of interactions among
CDK inhibitors and elements of the Rho
pathway allows the hypothesis that this
represents a key mechanism for coordi-
nating cytoskeletal functions with cell
division cycle. Importantly, the p27Kip1
activity on the Rho-dependent pathway is
definitely separated from its role in regu-
lating cell proliferation. As matter of fact,
wild-type p27Kip1 and a mutant unable
to interact with cyclins and CDKs were
equally potent in decreasing the number of actin stress fibers and
rescuing the migration defect of p27Kip1-null fibroblasts.
Disrupting these p27p27Kip1 activities might contribute to the disease
state, especially since recent evidence demonstrate that cancer cells, in
order to proliferate, do not require hyperactive CDKs Among these
studies on cell motility, it is worthy of note that McAllister et al,20
showed that a cytosolic pool of p27p27Kip1 stimulates Rac-dependent
migration in HepG2 cells and embryonic fibroblasts. This effect isG2 cells and embryonic fibroblasts. This effect is cells and embryonic fibroblasts. This effect is
due to the C-terminal domain in p27
CDK inhibitory activity.
In transformed cells, one could expect that the down-regulation
of p27Kip1 parallels to increased Rho activity and in inhibition of
migration. This is not what is observed. Indeed, cancer cells are
usually highly motile and invasive. Transformed cells have found
mechanisms to uncouple distinct effector pathways downstream of
Rho. For instance, in cells transformed by Ras, elevated levels of
Rho activity are selected to allow the downregulation of p21Cip1
and cell cycle progression, whereas high levels of ERK signaling
uncouple Rho from inducing actin stress fibers and focal adhesions
by down-regulating ROCK1 and ROCK2.61
On the other hand, it has been demonstrated that Ras activation
causes the cytosolic localization of p21Cip1 and the consequent inhi-
bition of ROCK activity. The last event uncouples RhoA from stress
p27Kip1 and was independent of
COntROl OF p27Kip1 COntent
The activities of p27Kip1 are controlled by its concentration, phos-
phorylation status, subcellular localization, and interactions with
other cellular complexes and assemblies. All these processes are clearly
interconnected in that the intrinsic efficiency of one event modulates
the efficiency of the other processes.
We will try to dissect the major events of p27Kip1 metabolism,
taking in mind that they are only few pieces of an incomplete puzzle,
as yet. Moreover, it is to underline that although excellent studies
have been performed on p27Kip1 metabolism, most results reported
in Literature are context-specific and cannot be employed for devel-
oping an integrated general picture.
Control of p27Kip1 gene expression. Although it is well established
that the control of p27Kip1 level is mostly due to the extent of
degradation rate, several reports describe the occurrence of a tran-
scriptional modulation of the CDKN1B gene.
Figure 2. Phosphorylatable p27Kip1 residues and their presumed protein kinases. The scheme reports the
residues of p27Kip1 that are subject to phosphorylation. In the boxes are reported the protein kinase(s)
that have been suggested to catalyze the reaction(s). In addition, the sequences of the protein involved
in the nucleus/cytosol shuttling are shown.
he scheme reports the
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model (serine 10-to-alanine homozygous mice) reevaluated the role
of serine 10 phosphorylation in nuclear exit.100 Given the complexity
and variety of models used to study the functional significance of
this phosphorylation site, further investigations will be needed to
discriminate between these two roles. However, again, it is possible
that the same modification plays different roles in distinct contexts.
p27Kip1 post‑synthetic modifications. The analysis of p27Kip1 by
2D electrophoresis has definitely demonstrated that the protein is
p27Kip1 Metabolism, An Intricate Web
Particularly, the following transcription factors have been reported
to be involved in CDKN1B transcriptional control: members of the
Forkhead box O (FOXO) transcription factor family,75-81 E2F1,82
c-Myc (or c-Myc/Max complex),83,84 Sp185 and menin.86
However, the structure and function of p27Kip1 promoter has
been scarcely investigated and few information are available on the
precise interaction of the above reported transcription factors with
the CKI promoter. The FOXO family of transcription factors repre-
sents the only exception. As matter of fact, the control of p27Kip1
RNA synthesis has been demonstrated to mediate cell cycle entry
when members of the FOXO family are inactivated by PI3K and its members of the FOXO family are inactivated by PI3K and its
downstream effector, protein kinase B.75,79
p27Kip1 localization and transport. As described before, p27Kip1
protein contains signals for both nucleus entry and exit. Regulation of
sub cellular localization is as important as the control mechanism of
p27Kip1 function and metabolism, since spatially separating p27
from cyclin-CDK complexes obviously allows cell cycle progression.
Mechanisms that control the nuclear-cytosol shuttling or the import
of p27p27Kip1 from the cytoplasm to the nucleus have been discovered,
and include phosphorylations of p27
phosphorylation of threonine 157 or 198 (or 197 in mouse) by Akt/
PKB, for example, inhibits nuclear import of p27
the accumulation of p27p27Kip1 in the cytoplasm and inhibition of G1
arrest.87-93 It is interesting that threonine 157, located in the human
p27Kip1 NLS, is absent in the mouse protein. However, in literature
exist examples of key residues subject to post-synthetic modification
and not conserved among different mammals. When threonine 157
or 198 are phosphorylated, a recognition domain for the 14-3-3
proteins is generated which competes with the importin-a binding
and leads to p27 p27Kip1 accumulation in the cytoplasm.94
Very recently, the phosphorylation of some tyrosines has also been
implicated in regulating the subcellular localization of p27
response to granulocyte colony-stimulating factor (G-CSF).95
Notwithstanding the importance of the phosphorylations described
above, p27 p27Kip1 phosphorylation on serine 10 seems to play a pivotal
role in the exit of the protein from the nucleus. The nuclear-cyto-
plasmic shuttling of p27p27Kip1 upon serine 10 phosphorylation requires
the binding of p27 p27Kip1 with CRM1/exportin.29,96 Interestingly,
serine 10 is not located in the putative NES.29 p27
shown to exit from the nucleus after its association with the signalo-
some component Jab1, which interacts with CRM1,97 and with the
nuclear pore associated protein mNPAP60 through arginine 90.30
How these interactions are related to the p27
status is not known. Serine 10 phosphorylation also causes the elon-
gation of the CKI half-life.98 To distinguish between roles of serine
10 phosphorylation in the stabilization of p27
nuclear-cytoplasmic shuttling of p27p27Kip1 on the other, Kotake et al.,
generated serine 10-to-alanine homozygous mice.99 While nuclear
export of p27p27Kip1 occurred normally in the lymphocytes of these
mice, the stability of p27 p27Kip1 was reduced in G0 phase, but not in S
phase, when compared to wild type.99 Therefore, these results suggest
that the unique role of serine 10 phosphorylation is the stabilization
of the CKI in the G0 phase. Subsequently, Besson et al. by a similar
p27Kip1 on different sites. The
p27Kip1, resulting in
p27Kip1 has also been
p27Kip1 on one hand or in
phosphorylated at multiple sites. It is generally accepted that these
post-synthetic modifications are phosphorylations on the threonine
and serine residues. However, a number of papers has also recently
reported the phosphorylation of p27Kip1 on tyrosine residues. The
occurrence of the tyrosine modifications has not yet conclusively
confirmed and its importance is not defined. Following, we will
describe each reported phosphorylation and then, we will try to gain
Phosphorylation on Serine 10. The phosphorylation of p27Kip1
on serine 10 was reported for the first time by the Nakayama’s group
in 200098 and confirmed by numerous studies. It is estimated that
70–75% of phosphate incorporation occurs on this residue that
represents the most abundant post-translational p27
tion. On the other hand, as discussed above, the physiological role(s)
of this protein phosphorylation is still not completely clarified.
A major confusion exists about the kinase(s) responsible for
serine 10 p27p27Kip1 phosphorylation. At least four enzymes have
been supposed to catalyze the reaction. These include MAPK,
human kinase interacting stathmin (hKis),
Dirk1B.103 An hypothesis that might explain the large number of
enzymes able to phosphorylate the CKI on Ser10 is that they act
during diverse processes and in different phenotypes. It is difficult to
analyze critically the data on these kinases as the methods for their
identification were not based on classical procedures of biochem-
ical purification. Moreover, some of the experimental approaches
employed in these studies, including the CKI phosphorylation in
vitro and the positive or negative handling of kinases by transfection,
are not fully convincing. In addition, the use of p27Kip1 mutated in
specific residues is unpredictable since the mutated proteins might
have altered structural features (particularly because the CKI is an
intrinsically disordered protein) that can interfere with its recogni-
tion and interaction with other cellular components.
hKis has been identified by Bohem and colleagues102 as putative
Ser10p27Kip1 kinase by a two hybrid screen analysis employing the
C-terminus of p27Kip1 as bait.102 The enzyme activation causes: i)
p27Kip1 cytosolic relocalization and subsequent degradation, and ii)
the activation of cell proliferation. Thus, hKis activity seems mostly
related to p27Kip1 removal and S entry from G G1/G G0 phases.102
However, the analysis of the various isoforms of the p27Kip1 suggests
that the CKI is continuously phosphorylated in serine 10 during
G1->� G G0 transition, a period of cell cycle when hKis activity is low.
Mirk/dyrk1B kinase also phosphorylates p27Kip1 at Ser10 and
increases during the entry in G G0 phase.103 Thus, it might be consid-
ered the enzyme that is required for phosphorylating the protein
when hKis is not operative. However, the distribution of the enzyme
is mostly confined to muscle, while S10 phosphorylated p27Kip1 is
ubiquitous. Furthermore, the enzyme is active in G G0, while in this
phase a scarce phosphorylation of p27Kip1 has been reported.99
The importance of AKT in the phosphorylation of p27Kip1
appears quite complex. Initially, the enzyme has been suggested
to modify the CKI in Thr157 or Thr198,87-94 but a recent paper
suggests that the kinase phosphorylates p27Kip1 mostly in serine 10
and then, in additional residue(s).104 In the same paper, the capability
of AKT to modify p27Kip1 in Thr157 or 198 was also questioned.104
If this study will be further confirmed, the mechanism by which
AKT causes the cytosolic accumulation of p27Kip1 by inducing the
binding of the CKI with the 14-3-3 protein should be revisited. It is
to underline that a strict relationship interconnects AKT and PTEN
activities, and that the phosphatase has been recently identified in
the nuclear compartment. Therefore, the effect of the PI3K/AKT
It is estimated that
. These include MAPK,101
(hKis),102 AKT/PKB and Mirk/
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when the CKI was phosphorylated in tyrosine.95 The activation of
the receptor (by G-CSF addition) results into a down-regulation
of p27Kip1 tyrosine phosphorylation, and reduced interaction with
CDK4, without any effect on CDK2 binding. Similarly, phosphor-
ylation of p27Kip1 by Abl increased the CKI affinity for CDK4 while
decreasing that for CDK2.
Subsequently, Grimmler et al.113 reported that the nonreceptor
tyrosine kinase Lyn binds to p27Kip1 and phosphorylates it on
p27Kip1 Metabolism, An Intricate Web
pathway on p27Kip1 requires additional studies. Interestingly, AKT
also regulates the expression of the CKI gene by negatively acting on
FOXO transcription factors.
The activation of Erk1/2 causes a p27Kip1 level decrease by
inducing the translocation of the protein from the nucleus to the
cytoplasm, followed by the CKI degradation. However, several find-
ings indicate that the effect is not due to a direct phosphorylation,
although in vitro experiments showed that activated Erk1/2 might
add a phosphate on serine 10.
In brief, the addition of a phosphate on serine 10 is undoubtedly
the major post-synthetic modification of p27Kip1, but the identifica-
tion of enzyme(s) responsible for this phosphorylation is still fading.
Phosphorylation on Threonine 187. The phosphorylation on
threonine 187 represents the only post-synthetic modification consis-
tently demonstrated and accepted. The phosphorylation is catalyzed
by the cyclin E/A-CDK2 complex and cyclin B-CDK1.105,106
Definitely, threonine 187 phosphorylation is required for the
binding of p27Kip1 to Skp2, the F-box protein component of an
SCF ubiquitin ligase (E3) complex, which acts in the p27Kip1
ubiquitinylation107-109 and its subsequent degradation by the ubiq-
uitin-proteasome system.110,111 Interstingly, although the binding
between cyclin E/A-CDK2 occurs at the N-terminus of p27Kip1,
the substrate residue is at the C-terminus. It is improbable that this
should allow the modified CKI C-terminus to interact with other
proteins even if the p27Kip1 is bound to the cyclin E/A-CDK2 complex.
Phosphorylation on Threonine 157 and/or Threonine 197/198. It It
is widely accepted that p27p27Kip1 is phosphorylated by activated AKT/
PKB (see also above). This effect directly connects the PI3K/AKT
pathway with the cell cycle by-passing more complex and indirect
biochemical connection.87-94 A number of reports have demon-
strated that AKT phosphorylates human p27
and/or 198. Moreover, specific antibodies against p27
phosphothreonine 157 have been prepared and used for confirming
the presence of the modified CKI in vivo.87-89
The activity of AKT/PKB on p27
possibility of the protein to be incorporated into the nucleus, thus
preventing the capability of CKI to act as growth inhibitor. Moreover,
the cytosolic increase of the protein might interfere with the normal
structure of the cytoskeleton and increase the metastatic capability of
the cells. In turn, this could explain the accumulation of p27
the cytosol of aggressive cancers. However, this well definite picture
has been modified by the above reported study suggesting that AKT
phosphorylates the CKI on serine 10.
As discussed above, a different mechanism of p27
rylation by AKT has been recently proposed, in that it has been
suggested that PKB initially modifies the CKI on serine 10 and,
secondary to this phosphorylation, it is able to modify p27
Phosphorylation on Tyrosine Residues. Several groups have
recently described the phosphorylation of p27Kip1 on tyrosine resi-
dues.95,112,113 Particularly, Kardinal and colleagues95 reported that in
NB4 cells (a cell line established from a human acute promielocytic
leukemia) p27Kip1 interacts with the G-CSF receptor and Grb2
p27Kip1 on threonine 157
p27Kip1 should hamper the
Several groups have
tyrosine 88. Similarly, The Abl phosphorylates p27Kip1 on tyrosine
88 and to a lesser extent on tyrosine 89.
The CKI modification causes a diminished ability of the protein
to inhibit CDK2, by affecting, in a complex way, the interaction
with the kinase-cyclin complex. Therefore, while the phosphotyro-
sine p27Kip1 does not inhibit CDK2, it can efficaciously bind the
kinase and be phosphorylated on threonine 187. Accordingly, the
phosphorylation on tyrosine residues probably results in an increased
instability of p27Kip1. Conversely, a mutant p27Kip1 form where
tyrosine 88 was substituted by a phenylalanine displayed an increased
stability in cells.
Finally, Chu and collagues112 analyzed breast cancer samples and
observed a strict correlation between activation of Src kinase and low
levels of p27Kip1 levels. In addition, in vitro p27Kip1 phosphorylated
on tyrosine 74 and 88 by active kinase shows a decreased binding to
CDK2 and a scarce inhibition of the kinase activity, in comparison
with the unmodified p27Kip1. In vivo, the functional inactivation of
Src by different strategies decreases the tyrosine phosphorylation of
p27Kip1 and upregulates the phosphorylation of threonine 187 along
with the protein stability of p27Kip1.
However, although these data are extremely interesting and might
represent a connection between the activation of tyrosine kinases
modulated by growth factors and cell cycle control, the results were
limited to experimental conditions where the proteins have been
increased by forced expression.
Other post-synthetic modifications. It has been reported that
p27Kip1 is phosphorylated in residues other than those above
reported. In particular, the phosphorylation in serine 178 has been
described.114 However, its role and function is, at present, scarcely
characterized. Two additional findings can be reported, First, experi-
ment of transfection, performed in our laboratory, with p27Kip1
contemporaneously mutated in serine 10, threonine 187 and serine
178 showed, by 2D analysis, the occurrence of several phosphorylated
signals (not showed). Second, bidimensional analyses performed in
our laboratory (and confirmed by other groups) showed the occur-
rence of a p27Kip1 isoform with a pI not due to a phosphorylation.115
Thus, in a future, we would expect additional p27Kip1 post-synthetic
p27Kip1 removal processes. The complexity of the CKI removal The complexity of the CKI removal
is extreme and we will not discuss it in this paper in details since it is
in part out of our aim.
In the nuclear compartment, threonine 187 phosphorylation
is required for the binding of p27Kip1 to Skp2, the F-box protein
component of an SCF ubiquitin ligase (E3) complex, which induces
p27Kip1 ubiquitinylation107-109 and its subsequent degradation by
the ubiquitin-proteasome system.110,111 However, a series of discrep-
ancies, including the interval of Skp2 expression and its cellular
distribution compared to p27Kip1 degradation, has led to envision
other degradation modes for the CKI. Indeed, while the level of
p27Kip1, high in G0, decreases rapidly upon entry in G1, Skp2 is
mostly expressed at the G1-S transition.116 Furthermore, Skp2 has a
peculiar nuclear localization,117 whereas p27Kip1 is exported from the
nucleus to the cytoplasm to allow G1->�S passage. Recently, an alter-
native ubiquitin ligase for p27Kip1 has been identified. KPC (Kip1
ubiquitylation-promoting complex), constituted of KPC1 and KPC2
subunits that act cooperatively, is responsible for p27Kip1 degradation
at the transition from G0 to G1.118-120
Additionally, the level of p27Kip1 can be regulated by alternative
proteolytic pathways following nuclear export of p27Kip1. Calpain
protease, for example, can degrade p27Kip1 in response to the MAP
It has been reported that
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©2007 LANDES BIOSCIENCE. DO NOT DISTRIBUTE.
into specific phenotype(s).
A final point needs to be considered in all the studies on p27p27Kip1.
The protein belongs to the class of intrinsically unfolded proteins.
Such proteins are frequently involved in some of the most important
regulatory functions in the cell, and the lack of intrinsic structure is
relieved when the protein binds to its target molecule. The structure
absence confers functional advantages to these proteins, including
the ability to bind several different cellular targets. The feature
p27Kip1 Metabolism, An Intricate Web
kinase signaling pathway.121,122 Finally, it has been recent proposed
the occurrence of additional mechanisms of the CKI removal, mostly
on the basis of indirect evidences, but their presence and molecular
mechanisms remain to be confirmed and clarified.103
A BRieF COnClusiOn
The apparent inconsistency of some data on p27Kip1 make
unthinkable to propose an unitary scheme of the CKI metabolism.
Probably, attempts of identifying stringent and general mechanisms
are erroneous by nature, since the processes investigated strongly
depend on the tissue context (or cell phenotype) and specific func-
tional status. Thus, the conflicting data in Literature should not be
simply ascribed to incorrect experimental approaches, but to the
inherent biological complexity.
Three critical aspects, however, seem to emerge: (1) p27Kip1 is a
complex phosphoprotein containing at least 6–8 phosphorylatable
residues (Ser10, Tyr57, Tyr88, Tyr89, Thr157, Ser178, Thr187 and
Thr198); (2) the cellular localization of the protein strongly influ-
ences its function and, possibly, its folding, and (3) the natively
disordered structure of p27Kip1 facilitates its interaction with several
distinct proteins and might be subjected to remarkable alteration
by different pattern of phosphorylation (or other post-synthetic
The number of kinases responsible for the phosphorylation of
p27Kip1 is at present undetermined. Except for the role of CDK2 in
modifying threonine 187, that is consistently identified, the enzymes
modifying the other phosphorylatable residues have been only puta-
tively characterized. However, it seems important to underline that
a number of reports converge on PI3K/AKT/PTEN pathway as a
pivotal watchdog of p27Kip1 level and localization, although whether
and when AKT directly modifies the CKI is not definitely established.
In the context of phosphorylation modifications, it is remarkable the
absence of studies on the possibility that some phosphoisoforms of
p27Kip1 might be dephosphorylated and, in a positive case, on the
protein phosphatase(s) responsible for this reaction.
An additional unresolved question is the importance of phos-
phorylation on serine 10 in the stability of the protein and/or in its
shuttling nucleus to cytosol. The accumulation of data in different
well-characterized experimental models might be of great aid in
defining the apparent discrepancies.
It is not surprising that the meaning of the changes of the
CKI localization is not completely clarified. When entering in a
different cellular territory (nucleus or cytosol), p27
to completely modify its function. In the nucleus, the protein is a
tumor suppressor while in the cytosol it acts as a dominant oncogene
endowed with a pro-metastatic capabilty. Accordingly, the distinct
activities require different removal activities, as clearly demon-
strated. However, the importance of phosphorylation, if it exists,
in the cytosol removal has not been completely clarified. The effect
of p27p27Kip1 on cell movement is also a very exciting aspect, but the
absence of this activity in p27p27Kip1 null mice does not seem to result
p27Kip1 is supposed
also allows precise control over binding process and provides a
simple mechanism for inducibility by phosphorylation or by other
post-synthetic changes. These in vivo advantages, on the other hand,
make complicated the evaluation of functional studies based on the
forced overexpression of natively unfolded proteins (either wild-type
or mutants) and/or of its putative targets. Similar difficulties exist
in identifying putative kinases of intrinsically disordered proteins.
All these problems might explain the difficulties in conciliating the
frequent controversial data on p27Kip1.
Finally, the amazing complexity of p27Kip1 metabolism makes
the identification of novel pieces of its puzzling physiology only new
starting points for unexpected possibilities and further debates and
rarely, albeit partial, definite conclusions.
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