From the Friedrich Miescher-Institut, P. O. Box 2543, 4002 Basel, Switzerland
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ABSTRACT |
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We have cloned human protein kinase B Three members of the protein kinase B
(PKB)1 subfamily of
second-messenger regulated serine/threonine protein kinases have been
identified and termed Phosphorylation of PKB Only a few studies have been reported on the third member of the PKB
family, PKB Cloning of Human HA-PKB Northern Blot Analysis--
Human adult and fetal multiple
tissue Northern blots (CLONTECH) were hybridized
with a 825-bp fragment encoding amino acids 110-384 of PKB Cell Culture, Immunoprecipitation, in Vitro Kinase Assays, and
Immunoblot Analysis--
Human embryonic kidney (HEK) 293 cells were
maintained and transfected by a modified calcium phosphate method as
described previously (15, 28). Stimulation was for 5 min with 0.2 mM pervanadate (7) or for 15 min with 500 nM
insulin (Boehringer Mannheim). Pretreatment with the PI3K inhibitor
wortmannin (200 nM; gift of Dr. Markus Thelen, Theodor
Kocher Institute, Bern, Switzerland) was for 15 min. Cells were
extracted and HA-PKB Screening of several human cDNA libraries led to the isolation
of 12 clones encoding partial and overlapping sections of the open
reading frame of human PKB (PKB
)
and found that it contains two regulatory phosphorylation sites,
Thr305 and Ser472, which correspond to
Thr308 and Ser473 of PKB
. Thus it differs
significantly from the previously published rat PKB
. We have also
isolated a similar clone from a mouse cDNA library. In human
tissues, PKB
is widely expressed as two transcripts. A mutational
analysis of the two regulatory sites of human PKB
showed that
phosphorylation of both sites, occurring in a phosphoinositide 3-kinase-dependent manner, is required for full activity.
Our results suggest that the two phosphorylation sites act in concert to produce full activation of PKB
, similar to PKB
. This contrasts with rat PKB
, which is thought to be regulated by
3-phosphoinositide-dependent protein kinase 1 alone.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
,
, and
(1-4). The isoforms are homologous, particularly in regions encoding the N-terminal pleckstrin homology (PH) and the catalytic domains. PKBs are activated by phosphorylation events occurring in response to phosphoinositide 3-kinase (PI3K) signaling (5-8). PI3K phosphorylates membrane inositol
phospholipids, generating the second messengers phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate (reviewed
in Ref. 9), which have been shown to bind to the PH domain of PKB (10,
11). The current model of PKB activation proposes recruitment of the
enzyme to the membrane by 3'-phosphorylated phosphoinositides, where
phosphorylation of the regulatory sites of PKB by the upstream kinases
occurs (12-14).
/
occurs on two regulatory sites,
Thr308/309 in the activation loop in the catalytic domain
and Ser473/474 in the C-terminal domain (15, 16). The
upstream kinase, which phosphorylates PKB
at the activation loop
site Thr308, has been cloned and termed
3-phosphoinositide-dependent protein kinase 1 (PDK1; Refs.
17-20). PDK1 phosphorylates not only PKB
, but also equivalent sites
in the p70 ribosomal S6 kinase (21, 22), protein kinase A (23), and
protein kinase C (24). The upstream kinase phosphorylating the second
regulatory site of PKB
/
, Ser473/474, has not been
identified yet, but a recent report implies a role for the
integrin-linked kinase (ILK-1), a serine/threonine protein kinase
(25).
, and these all involved a clone originating from a rat
brain cDNA library (4, 26). A major feature distinguishing rat
PKB
from the otherwise very similar
and
isoforms is the C
terminus, which is truncated by 23 amino acids and lacks
Ser473/474, one of the two phosphorylation sites essential
for activation of PKB
and
(15, 16). Consequently, it has been
suggested that rat PKB
activation depends solely on the upstream
kinase PDK1. We now report the cloning and characterization of human PKB
. This isoform differs significantly from the rat enzyme in that
it contains a C-terminal domain similar to PKB
/
, with a putative
second regulatory phosphorylation site at Ser472.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
and Mutant Isoforms--
A 525-bp PCR
product corresponding to nucleotides 815-1340 of the rat PKB
sequence (4) was amplified from mouse brain cDNA and used to screen
several different human cDNA libraries. Twelve overlapping clones
were isolated and assembled to a cDNA encoding amino acids 16-479
of human PKB
. This cDNA was repaired by PCR-mediated addition of
a hemagglutinin (HA) tag and the missing N-terminal amino acids deduced
from the rat PKB
sequence and ligated as a
KpnI/XbaI fragment into the pCMV5 eucaryotic
expression vector (27). Subsequently, the 5' end of PKB
was
amplified by 5'-rapid amplification of cDNA ends from human brain
cDNA. A mouse brain cDNA library screened with the same probe
yielded 13 overlapping clones which could be assembled into a cDNA
encoding the entire reading frame of mouse PKB
. Mutations in
HA-PKB
(HA-PKB
T305A and HA-PKB
T305D) were done by Quikchange
(Stratagene) or with mutagenizing 3' primers (HA-PKB
S472A and
HA-PKB
S472D). HA-
PHPKB
was obtained by PCR with a primer
encoding the HA-tag and amino acids 119-126. All PCR-cloned constructs
were verified by DNA sequencing.
according to the manufacturer's instructions.
activity determined exactly as described in
Ref. 28. Western blot analysis was performed as described before (15)
and developed with the polyclonal phospho-specific Ser473
antibody (1:1000, New England Biolabs), an alkaline phosphatase (AP)-coupled goat-anti mouse IgG secondary antibody (1:2000, Sigma), and alkaline phosphatase color development reagents (Boehringer Mannheim).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
, and the cDNA was assembled as described under "Experimental Procedures." The 479-residue amino acid sequence of human PKB
is presented in Fig.
1, as an alignment with human PKB
,
PKB
, mouse PKB
(see below), and with the C-terminal domain of rat
PKB
. Human PKB
is 83% identical to PKB
, 78% identical to
PKB
, and 99% identical to rat PKB
(two changes in 451 amino acids and a different C terminus), indicating that we have isolated the
authentic human PKB
isoform and not a PKB
or PKB
variant. Moreover, we cloned a similar PKB
from a mouse brain cDNA
library, demonstrating that this isoform is not restricted to one
species. Human and mouse PKB
were found to be more than 99%
identical (2 amino acid changes in 479). The major characteristic
distinguishing human and mouse PKB
from the rat isoform is the
presence of a C-terminal domain similar to PKB
and
, containing a
second putative regulatory phosphorylation site at Ser472
(marked with an asterisk in Fig. 1). To ascertain whether
the human PKB
cDNA corresponded to the major mRNA species,
we performed 3'-rapid amplification of cDNA ends and found that all
15 clones sequenced, which spanned the C terminus of the protein
contained the Ser472 domain. However, human and rat PKB
diverge in amino acid sequence precisely at a site where an exon
boundary has been mapped for the mouse PKB
gene (29). Thus, it is
possible that the published rat cDNA sequence constitutes a minor
splice variant of PKB
or a partially processed mRNA.
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Fig. 1.
Sequence alignment of human
PKB , PKB
,
PKB
, mouse PKB
, and
the C terminus of rat PKB
. Shown are the
amino acid sequences of human PKB
(1), PKB
(3), and PKB
, of
mouse PKB
, and the C-terminal domain of rat PKB
(4). The
numbering refers to PKB
. The regulatory phosphorylation sites
(Thr308 and Ser473 in PKB
) are indicated
with an asterisk.
To assess the tissue distribution of transcripts encoding PKB, we
used an isoform-specific radiolabeled cDNA fragment to probe two
human multiple tissue Northern blots. Two equally expressed transcripts
of 8.5 and 6.5 kilobases were detected in all tissues tested, with
highest levels found in adult brain, lung, and kidney and very low
levels in heart and liver (Fig.
2A). Two transcripts of
similar size were detected in fetal tissues (Fig. 2B), with high levels found in heart, brain, and liver, but none in the kidney.
This observation, and the size of the transcripts, which are much
larger than the 3.2-3.4-kilobases transcripts of
PKB
/
,2 indicated the
presence of long untranslated regions, and thus the possibility of
developmental regulation of expression or post-transcriptional modifications affecting mRNA stability.
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In contrast to the rat enzyme, human PKB contains two predicted
regulatory phosphorylation sites, Thr305 in the activation
loop, and Ser472 in the C-terminal domain, as does
PKB
/
. To determine the importance of these two residues, we
mutated them to alanine which cannot be phosphorylated, or to aspartate
to mimic the phosphorylated state, and assayed HA-PKB
kinase
activity following transient transfection and stimulation with the
insulin mimetic compound pervanadate (Fig.
3). The results presented here show that
wild type HA-PKB
, which had a low basal activity, could be
stimulated 67-fold by pervanadate treatment; furthermore, mutation of
Thr305 to alanine completely ablated activation. No
activity above basal levels was observed for HA-PKB
T305A, and the
same was true for the double mutant HA-PKB
T305A,S472A. On the other
hand, mutation of the C-terminal regulatory site (HA-PKB
S472A)
reduced but did not abolish activation by pervanadate (10-fold). We
also tested the effects of aspartate mutations, since in the case of
PKB
, a double aspartate mutant was constitutively active (15).
However, PKB
was not active above basal levels upon mutation of the
activation loop site to aspartate (HA-PKB
T305D and
HA-PKB
T305D,S472D) and, furthermore, could not be stimulated by
pervanadate treatment. Thus the aspartic acid moiety could not
substitute for the phosphorylated threonine residue. Again, mutation of
Ser472 to aspartate (HA-PKB
S472D) resulted in a protein
that could still be activated by pervanadate (35-fold), albeit to a
lesser extent than the wild type. These results establish that the
phosphorylation site Thr305 in the activation loop is
absolutely necessary for activation of PKB
, with conformational
constraints around the active site apparently so stringent that
substitution by a negatively charged residue is not tolerated.
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To determine the role of the C-terminal Ser472 in
regulation of human PKB, we tested whether activation of HA-PKB
,
HA-PKB
S472A, and HA-PKB
S472D by insulin was sensitive to
inhibition of PI3K. Fig. 4A
depicts the results of a representative experiment, which show that
insulin activated HA-PKB
, HA-PKB
S472D, and, to a lesser extent,
HA-PKB
S472A. This stimulation was dependent on the activity of PI3K,
since pretreatment of transfected cells with the PI3K inhibitor
wortmannin inhibited activation by insulin. Furthermore, we subjected
the immunoprecipitated proteins to Western blot analysis with an
antibody generated specifically against the phosphorylated Ser473 peptide of PKB
(Fig. 4A, inset). The
antibody cross-reacted with HA-PKB
only upon stimulation with
insulin, and phosphorylation of Ser472 was prevented by
wortmannin. Since Ser472 was mutated in HA-PKB
S472A and
HA-PKB
S472D and could not be phosphorylated, we concluded that the
wortmannin-sensitive, insulin-stimulated activity of these proteins was
entirely due to phosphorylation at Thr305, dependent on the
presence of 3-phosphorylated phospholipids.
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In this analysis, we also included PKB constructs lacking the
N-terminal PH domain. In the basal state, this domain is thought to
restrict access to the phosphorylation site in the activation loop,
thus leaving Thr305 more accessible to phosphorylation by
upstream kinases when it is removed. The proteins lacking the PH domain
now presented a different picture (Fig. 4B): HA-
PHPKB
was maximally activated under basal conditions and could not be
stimulated further by insulin treatment. This contrasts with results
obtained for
PHPKB
, shown to be activated by insulin (6).
Furthermore, pretreatment of the cells with wortmannin led to a
reduction in activity of HA-
PHPKB
, indicating that it was still a
target for PI3K-dependent phosphorylation.
HA-
PHPKB
S472A activity was comparable with that of
wortmannin-treated HA-
PHPKB
and was not responsive to insulin or
wortmannin. In contrast, HA-
PHPKB
S472D was again fully active in
the absence of stimulation but, unlike HA-
PHPKB
, was not
inhibited by wortmannin. The Western blot signals with the
phospho-specific Ser473 antibody correlated with the
activities observed (Fig. 4B, inset); HA-
PHPKB
was
strongly phosphorylated in extracts of unstimulated and
insulin-stimulated cells, but pretreatment with wortmannin reduced the
signal. We found previously that transiently transfected PDK1, the
upstream kinase phosphorylating Thr308 in PKB
, is active
in serum-starved HEK-293
cells.3 The present results
seem to indicate that removal of the PH domain causes a conformational
change of PKB
favorable to phosphorylation at Thr305, so
as to make it independent of PI3K activity. Basal activity of PI3K in
unstimulated cells allowed phosphorylation of HA-
PHPKB
by the
Ser473 kinase, resulting in full activation. Conversely,
inhibiting this basal PI3K activity by wortmannin treatment reduced
HA-
PHPKB
activity, probably due to the rapid action of
phosphatases on phosphorylated Ser472. Thus, HA-
PHPKB
is a model for studying phosphorylation of Ser472, the
second regulatory site of human PKB
, almost independent of
Thr305.
In summary, we report the cloning and characterization of human PKB,
a PKB isoform distinct from its rat counterpart in having two
regulatory phosphorylation sites, Thr305 and
Ser472, both of which are required for full activation of
the protein. Our results suggest markedly similar regulation mechanisms
for PKB
and the
and
isoforms, with both upstream kinases
phosphorylating the regulatory sites being sensitive to PI3K-derived
signals. Furthermore, we found a high abundance of PKB
mRNAs
encoding the C-terminal hydrophobic domain and have isolated a similar mouse PKB
, showing that this isoform is not restricted to humans. Taken together, we conclude that the truncated rat PKB
used in all
studies so far (26) probably constitutes a minor splice variant of
endogenous PKB
protein. The crucial question now emerging is that of
the specific roles of the three different PKB isoforms.
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ACKNOWLEDGEMENTS |
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We thank Drs. M. Andjelkovic, T. Millward, and P. King for comments on the manuscript; P. Mueller for oligonucleotide synthesis; and Dr. H. Angliker for DNA sequencing.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF124141 and AF124142.
To whom correspondence should be addressed: Friedrich
Miescher-Institut, P. O. Box 2543, CH-4002 Basel, Switzerland. Tel.: 41-61-697-40-46; Fax: 41-61-697-39-76; E-mail: hemmings{at}fmi.ch.
2 B. A. Hemmings, unpublished results.
3 M. Andjelkovic and D. Brodbeck, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: PKB, protein kinase B; PH, pleckstrin homology; PI3K, phosphoinositide 3-kinase; PDK1, 3-phosphoinositide-dependent protein kinase 1; HA, hemagglutinin; HEK, human embryonic kidney; bp, base pair; PCR, polymerase chain reaction.
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