MRC Protein Phosphorylation Unit, Department of Life Sciences, University
of Dundee, Dundee DD1 5EH, UK
*
Author for correspondence (e-mail:
d.r.alessi{at}dundee.ac.uk
)
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SUMMARY |
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Key words: Phosphoinositide 3-kinase, PDK1, AGC kinases, Docking sites, Phospho-specific antibodies, Insulin
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Introduction |
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![]() |
Identification of physiological substrates for PKB |
---|
|
Apart from the residues immediately surrounding the phosphorylation site on
a protein, there is increasing evidence that more distant residues can also
interact specifically with kinases, and these interactions are frequently
referred to as docking interactions/sites. In the case of MAP kinase family
members ERK1/ERK2, p38 and JNK, recent work shows that docking interactions
play crucial roles in enabling these kinases to phosphorylate specific
substrates (Holland and Cooper,
1999; Tanoue et al.,
2001
). Note that virtually all
of the characterised PKB substrates listed in
Table 1 physically interact
with PKB, but, thus far, the residues that mediate these interactions have not
been identified. PKB-docking sites, in addition to residues immediately
surrounding the phosphorylated serine/threonine residue, might also play a key
role in determining whether substrates are phosphorylated by PKB efficiently.
Thus, a protein that has a non-optimal PKB-phosphorylation motif could still
be a good physiological substrate if it can interact with PKB through a
high-affinity docking site.
![]() |
The role of PKB in promoting cancer progression |
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Mechanisms by which PKB can promote proliferation |
---|
|
PKB also regulates transcription of the p27 CDK inhibitor. Activation of
PKB or overexpression of constitutively active forms of this enzyme decreases
the cellular levels of p27, thereby promoting cell proliferation (Gesbert et
al., 2000; Graff et al.,
2000
; Sun et al.,
1999
). More recent studies
indicate PKB phosphorylates the forkhead transcription factor, which is
required for transcription of p27. Again phosphorylation results in its
binding to 14-3-3 proteins, which sequester it in the cytoplasm of cells, in
which location it cannot induce p27 (Medema et al.,
2000
; Nakamura et al.,
2000
).
Other key regulators of cell cycle progression are the cyclin D proteins
(D1, D2 and D3), which accumulate during G1 phase and are required
for the activity CDK4 and CDK6. The levels of cyclin D are controlled at the
levels of transcription, translation and protein stability. Many growth
factors promote transcription of this cell cycle regulator, and multiple
signal transduction pathways, including the ERK1/ERK2 pathway (Cheng et al.,
1998; Lavoie et al.,
1996
; Schwartz and Assoian,
2001
), are involved.
Initially, it was shown that a PI-3-kinase-dependent pathway also plays an
important role in this process (Klippel et al.,
1998
; Takuwa et al.,
1999
), and subsequent studies
have suggested PKB is involved (Crowder and Freeman,
1998
; Gille and Downward,
1999
). There is also evidence
that overexpression of PI 3-kinase and PKB in cells stimulates the rate of
translation of cyclin D (Muise-Helmericks et al.,
1998
). The transcriptional and
translational components that PKB phosphorylates to induce cyclin D expression
remain to be identified.
Another important mechanism by which cyclin D1 levels are regulated is by
translocation of cyclin D1 from the nucleus to the cytoplasm, where it is
degraded by proteosomes following its ubiquitination (Diehl et al.,
1998). A key step in promoting
this pathway is the phosphorylation of cyclin D1 at Thr286 by glycogen
synthase kinase 3 (GSK3), which promotes the interaction of cyclin D1 with the
nuclear exportin CRM1 (Alt et al.,
2000
). GSK3 was the first
substrate of PKB to be identified, and its phosphorylation by PKB at an
N-terminal non-catalytic residue is inhibitory (Cross et al.,
1995
). In principle,
therefore, activation of PKB should result in GSK3 inactivation and decreased
phosphorylation of cyclin D1 by GSK3 and therefore maintain high cyclin D1
levels in the nucleus. Indeed, overexpression of a constitutively active PKB
promotes stabilisation of cyclin D1 protein (Diehl et al.,
1998
). However, we believe
that this situation could be more complicated. Many of the substrates that
GSK3 phosphorylates, such as glycogen synthase and eIF2B, require a priming
phosphorylation at residue n+4 (where n is the site of phosphorylation) in
order to be phosphorylated by GSK3 efficiently. The molecular basis of this
effect has recently been elucidated by the finding that the kinase domain of
GSK3 possesses a phosphate-docking site that interacts with the primed
phosphorylation site on the substrate (Dajani et al.,
2001
; Frame et al.,
2001
). Phosphorylation of GSK3
by PKB results in binding of the phosphorylated N-terminal residues of GSK3 to
this docking motif and inhibits phosphorylation of these substrates by GSK3. A
group of GSK3 substrates, however, do not require a priming phosphorylation
site. These include components of the Wnt signalling pathway, such as axin and
ß-catenin. Cyclin D1 is likely to fall into the same category, given that
it lacks phosphorylatable residues at the n+4 position. If this is the case,
then phosphorylation of GSK3 by PKB might not necessarily affect the rate at
which GSK3 phosphorylates cyclin D, and thus PKB might promote stabilisation
of cyclin D through a non-GSK3-dependent mechanism. Further studies are
required to clarify this situation.
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Mechanisms by which PKB can inhibit apoptosis |
---|
PKB could directly phosphorylate and inhibit the caspase proteases
key executioners of apoptosis. Indeed several caspases possess putative
PKB-phosphorylation sites (Table
1). Significantly, the site in human caspase-9 (Ser196) that is
phosphorylated by PKB (Cardone et al.,
1998) is not conserved in
monkey and rodent homologues, making it unlikely that phosphorylation of
caspase-9 is a key mechanism regulating apoptosis. A more recently identified
substrate is the apoptosis signal-regulating kinase 1 (Ask1), which stimulates
MAP kinase kinases that activate the JNK and p38 MAP kinases. Overexpression
of activated PKB phosphorylates ASK1 at Ser83, resulting in inhibition of
Ask-1 activity and reduced JNK activity (Kim et al.,
2001
). In some situations JNK
can promote apoptosis; in this context, inactivation of ASK1 by PKB could
promote cell survival. PKB activation might inhibit apoptosis by promoting the
increased expression of survival molecules or the degradation of pro-apoptotic
molecules. There are no examples, thus far, of the latter but overexpression
of a constitutively active PKB in cells does induce transcription of Flip, an
inhibitor of caspase-8 (Panka et al.,
2001
) and the p21 CDK
inhibitor (Lawlor and Rotwein,
2000
; Mitsuuchi et al.,
2000
). The molecular
mechanisms by which this occurs remain uncharacterised.
PKB also phosphorylates and activates endothelial nitric oxide synthase,
thereby promoting angiogenesis (formation of new blood vessels). In tumours
this increases the supply of nutrients to cancer cells, thereby promoting
their survival (Snyder and Jaffrey,
1999). PKB activity is also
stimulated following hypoxia. This has been shown to result in the activation
of the hypoxia-inducible factor 1, a transcription factor that induces the
expression of several genes that promote angiogenesis (Zhong et al.,
2000
).
Inhibition of GSK3 following its phosphorylation by PKB has also been
suggested to play a role in inhibiting apoptosis in neuronal cells. This is
based on the finding that overexpression of dominant negative GSK3 or
overexpression of a GSK3 inhibitor protein promotes neuronal cell survival in
response to inhibitors of PI 3-kinase (Ding et al.,
2000). Furthermore, selective
and potent small-molecule inhibitors of GSK3 have recently been developed,
termed SB-415286 and SB-216763 (Coghlan et al.,
2000
), and these have also
been shown, together with lithium, a less specific GSK3 inhibitor, to protect
both central and peripheral nervous system neurons in culture from death
induced by reduced PI 3-kinase pathway activity (Cross et al.,
2000
). The proteins that GSK3
phosphorylates to induce apoptosis remain to be defined.
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Mechanisms by which PKB promotes insulin-signalling responses |
---|
Other data suggest that PKB phosphorylation and inactivation of GSK3 is
likely to stimulate the conversion of nutrients such as glucose and amino
acids to storage macromolecules (glycogen and protein) in skeletal muscle,
adipose tissue and liver (reviewed by Alessi,
2001). Kasuga and colleagues
have shown that PKB phosphorylates and activates the cAMP-phosphodiesterase 3B
in adipocytes (Kitamura et al.,
1999
). This reduces the levels
of cAMP and hence the activity of PKA, which antagonises some insulin
signalling events. Note that the Yaffe-Cantley algorithm indicates that the
site in PDE3B phosphorylated by PKB lies outside the top 5% of optimal
PKB-phosphorylation motifs in the database. This is probably due to the
presence of a serine residue, rather than a hydrophobic residue, following the
residue phosphorylated by PKB (Table
1). The cardiac-specific isoform of 6-phosphofructo 2-kinase is
also activated following its phosphorylation on two residues by PKB, and this
is thought to underlie the mechanism by which insulin stimulates glycolysis in
the heart. Another potential substrate for PKB is the insulin receptor
substrate 1 (IRS1) adaptor molecule. Its phosphorylation is proposed to
inhibit recruitment of PI 3-kinase to the membrane and may play a role in a
negative feedback loop shutting off PI 3-kinase activity following prolonged
insulin stimulation (Li et al.,
1999
). The sites on IRS1
phosphorylated by PKB have not been mapped; however, IRS1 contains five
potential PKB-phosphorylation sites: two high-stringency hits (Ser307 and Ser
527, Table 1) and three
low-stringency hits (Ser270, Ser 330 and Ser1101). Overexpression of PKB in
several insulin-responsive cell lines also stimulates the uptake of nutrients
such as glucose and amino acids, and induces gene expression normally mediated
by insulin (reviewed by Hajduch et al.,
2001
). The mechanisms by which
PKB mediates these effects remain unknown.
Many key discoveries in the insulin/PI 3-kinase/PKB signaling pathway have
been made in model organisms. For example, the finding that PKB regulates the
forkhead transcription factor was first established in Caenorhabditis
elegans (Paradis and Ruvkun,
1998). Inactivation of
components of the PI 3-kinase signaling pathway in Drosophila has
been shown to markedly reduce cell size. Recent data have identified a new
downstream component of the PI 3-kinase pathway in Drosophila, namely
the Tuberous Sclerosis Complex genes Tsc1 and Tsc2. Loss of
Tsc1 and Tsc2 expression results in increased cell size, and
conversely overexpression of these genes decreases cell size considerably.
Genetic evidence from these studies suggests that Tsc1 and
Tsc2 function to inhibit the insulin signaling pathway downstream of
PKB (Gao and Pan, 2001
; Potter
et al., 2001
; Tapon et al.,
2001
). Interestingly, there
are number of potential PKB-phosphorylation sites in human and
Drosophila TSC2 (but not TSC1), of which one is a high-stringency
site (Table 1).
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PKB as a therapeutic target for the treatment of cancer and diabetes |
---|
There is also interest in generating drugs that can activate PKB, which could potentially be used to trigger insulin-dependent processes for the treatment of diabetes. These compounds could also be used to promote survival and inhibit apoptosis of neuronal cells following a stroke. Although it is intrinsically much harder to develop an activator of a kinase than an inhibitor, in the case of PKB a drug that binds to the PH domain of PKB, instead of inhibiting PtdIns(3,4,5)P3 binding, might mimic PtdIns(3,4,5)P3 and enable PDK1 to phosphorylate and activate PKB in unstimulated cells. If PKB activators could be developed, an obvious potential side effect is cancer. Although this might be a problem for the treatment of diabetes, where it would be necessary to administer the drug over a long period of time, it is less of a problem for the treatment of strokes, for which these drugs might only be required for a relatively short time.
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Potential pitfalls in approaches used to study PKB function |
---|
Insulin and growth factors frequently induce a transient activation of PKB,
and it is doubtful that the overexpression of high levels of activated PKB
mutants in cells for hours or even days is physiological. To overcome this
problem, Richard Roth and colleagues have generated a conditionally active
version of PKB fused to the estrogen receptor, which is inactive when
expressed in cells but is activated within minutes of stimulation by
4-hydroxytamoxifen (Kohn et al.,
1998). Another concern is that
most of the constitutively active PKB mutants that have been employed are
forcibly attached to the plasma membrane by the addition of a
membrane-targeting motif. In contrast, endogenous PKB is likely to
phosphorylate most of its substrates either in the nucleus or cytoplasm of
cells and not at cell membranes. Thomas and colleagues have demonstrated that
the overexpression of a membrane-targeted PKB induces activation of S6K1,
whereas overexpression of a non-membrane-targeted PKB mutant that has
identical activity does not have this effect (Dufner et al.,
1999
). This demonstrates that
a membrane-targeted PKB can trigger non-physiological processes. There are two
forms of non-membrane-targeted constitutively active PKB that can be employed:
one is a PKB mutant in which both the Thr308 and Ser473 activating
phosphorylation sites are changed to aspartate residues (Alessi et al.,
1996a
), and the other is mutant
of PKB in which the hydrophobic motif of PKB is replaced with that found in
PKC-related kinase 2 (PRK2), which possesses a very high intrinsic affinity
for PDK1 (Biondi et al.,
2001
).
Unfortunately, no specific PKB inhibitors or cell lines that lack expression of all PKB isoforms have been developed. Without such tools we cannot rule out the possibility that the phosphorylation of even a potential PKB substrate protein, such as GSK3 or forkhead, is in fact not mediated by PKB but rather by another PI-3-kinase-activated protein kinase, such as SGK. Although dominant negative forms of PKB have been used extensively to dissect the signalling networks that are regulated by PKB, great caution must also be employed when one uses these reagents, because the mechanism by which a dominant negative PKB mutant is functioning in cells is not known. For example, dominant negative PKB might work by interacting with and inhibiting PDK1, which could affect the phosphorylation of other AGC kinases that it activates, such as SGK. Additionally, dominant negative PKB could also interact non-physiologically with a substrate of another PI-3-kinase-activated protein kinase when overexpressed in cells, and this could prevent this substrate from becoming phosphorylated by its natural upstream kinase.
Finally, analysis of phosphorylation sites of proteins in cells is
increasingly being performed using phospho-specific antibodies, many of which
are generated by commercial companies and, unfortunately, in most cases these
have not been adequately characterised. In our opinion an essential control to
be carried out with every immuno-blot shown in a paper using a
phospho-specific antibody is that the phosphopeptide antigen used to raise the
antibody, but not the dephosphopeptide antigen must be shown to prevent the
recognition of the phosphorylated protein by the antibody. This is also
important when using commercial polyclonal antibodies, because different
batches of antibodies are likely to be derived from different animals and
different bleeds. Thus the specificity of the antibody may vary. Unless the
companies selling phosphopeptide antibodies are willing to provide the
phospho-specific and dephospho-peptide immunogens as controls with each batch
of antibody, we recommend that these antibodies not be employed. Given that a
phospho-specific antibody can frequently crossreact with other phosphorylation
sites on a protein, it is also essential to demonstrate that mutation of the
phosphorylatable serine/threonine to alanine or valine abolishes recognition
of the protein by the antibody. Unfortunately, these simple but key controls
are not being performed routinely, and this can lead to unreliable results.
For example, a study by our colleagues in Dundee found that a commercially
produced phospho-specific antibody raised against one phosphorylation site on
Bad also crossreacts with another phosphorylation site on this protein. This
could potentially lead to inaccurate conclusions being obtained by other
groups who used this antibody (Lizcano et al.,
2000). It is also important to
bear in mind that one cannot establish stoichiometries of phosphorylation by
using phospho-specific antibodies. These reagents can readily detect trace
levels of phosphorylation of a substrate in vitro. Especially for in vitro
phosphorylation experiments, one must demonstrate that the substrate is
phosphorylated to a significant stoichiometry by using standard peptide
mapping procedures and not rely exclusively on the use of phospho-specific
antibodies to infer that a given substrate is becoming phosphorylated
significantly.
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Concluding remarks |
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ACKNOWLEDGMENTS |
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