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INTRACELLULAR SIGNAL SYSTEMS |
Departments of Medicine (D.L.D., A.G.-H.) and of Biochemistry and Molecular Genetics (A.G.-H.) Colorado Cancer Center University of Colorado Health Sciences Center Denver, Colorado 80262
Address all correspondence and requests for reprints to: Arthur Gutierrez-Hartmann, M.D., University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Box B-151, Denver, Colorado 80262. E-mail: dawn.duval@uchac.edu and a.gutierrez-hartmann{at}ucshc.edu
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Introduction |
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 Top Introduction References |
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B kinase (IKK), endothelial
nitric oxide synthase, the proapoptotic protein BAD, members of the
forkhead family of transcription factors including forkhead in
rhabdomyosarcoma, AFX, daf16, BRCA-1, cAMP response element binding
protein (CREB), and p300 (2, 3, 4, 5). An analysis of the AKT
substrate phosphorylation sites in these direct targets has revealed a
minimal consensus site consisting of RXRXXS/T (2).
Additionally, indirect mechanisms of activation by AKT have also been
identified, e.g. AKT activates cyclin D1 via glycogen
synthase kinase-3 (2) and AKT activates p54 Jun N-terminal
kinase (JNK) to phosphorylate Ets-2 at T72 (6). The
AKT-mediated regulation of transcription factor potency is of
particular interest because gene repertoires that control key
biological processes, such as cell survival, are likely to be
coordinately regulated.
In this issue of Endocrinology, Hayakawa et
al. (7) report that activation of the proximal rat
PRL (rPRL) promoter by PRL-releasing peptide (PrRP) occurs via a
pertussis toxin-sensitive GPCR and ß
-subunit, signaling to the
rPRL promoter in a pathway that involves PI3K, AKT, CREB, and Ets. This
report also demonstrates that insulin stimulation of the rPRL promoter
is partially mediated through a similar pathway involving PI3K, AKT,
CREB, and Ets. One of the more interesting aspects of this paper is the
observation that a hypothalamic factor that acts as a PRL secretagogue,
PrRP, also serves to activate the PI3K/AKT pathway, which is associated
with cell survival and growth. In this regard, PrRP may function not
only to control PRL secretion and gene transcription, but also to
maintain lactotroph cell number. Additionally, this report provides
another example of a GPCR activating the AKT pathway (2, 8). The identification of CREB and Ets factors as nuclear
effectors of the AKT pathway is consistent with previous reports
implicating these factors as either direct (CREB) or indirect (ETS)
targets of AKT (3, 6). However, the identification of CREB
as a key AKT mediator of rPRL promoter activity is surprising because
it has been shown by several groups that recombinant CREB fails to bind
to the proximal rPRL promoter (9, 10), including the
current report by Hayakawa et al. (7). In this
report (7), they show that two different forms of
dominant-negative CREB (M1-CREB and K-CREB) are each able to partially
diminish the PrRP and insulin responses of the rPRL promoter, whereas
they completely inhibited the rPRL promoter activation mediated by a
constitutively active AKT. Given that CREB fails to bind to the
proximal rPRL promoter, one possible explanation of the data presented
here is that the dominant-negative CREBs may be titrating a limiting
transcription co-factor. Nevertheless, the precise role of CREB in
AKT-mediated regulation of the rPRL promoter remains unclear. By
contrast, Ets factors have been shown to be indirect targets of AKT
(6), and several members of the ETS family clearly bind to
the rPRL promoter and activate it in response to oncogenic Ras, growth
factors, and GPCR ligands (11, 12, 13, 14, 15, 16, 17). In this regard,
it is noteworthy that etsZ, a dominant-negative Ets, is able to
completely block rPRL promoter stimulation mediated by PrRP
(15), insulin, and dominant-active AKT (7).
This complete block of the PrRP and constitutively active AKT responses
by etsZ argues that an ETS family member is playing a very important
role in the regulation of the rPRL promoter by these signaling
molecules. Unfortunately, the authors do not map the cis-acting
elements of the rPRL promoter that are required for the PrRP and AKT
responses, and thus it remains to be determined whether the PrRP and
AKT sites colocalize and if they map to an Ets binding site.
So how might AKT target ETS factors? There have been two
recent reports showing that AKT modulates the phosphorylation of Ets
factors, the hematopoeitic-specific PU.1 (5) and Ets-2
(6). AKT activation mediated through PU.1 has been mapped
to an acid rich region (AA 33–74) of its transcriptional activation
domain. Point mutation of S41 blocks AKT-mediated induction of the
E3'-enhancer, suggesting that phosphorylation of this site is
responsible for activation. This serine residue lies within an SXXXS
motif, which does not match the AKT consensus site, but rather is
homologous to the consensus phosphorylation site for IKKs. Although IKK
has not been directly implicated in the activation of PU.1, previous
studies have identified IKK as a direct target of AKT (2).
In the case of Ets-2, Smith et al. (6) show
that AKT mediates the phosphorylation of Ets-2 at a proline-directed
phosphorylation site (PLLT72P) in an indirect
manner. The targeting of AKT to the T72 MAPK phosphorylation site
provided evidence that AKT was unlikely to directly phosphorylate
Ets-2. Rather, AKT activates JNK, which then directly phosphorylates
Ets-2 at T72. Taken together, these data explain the enhanced Ets-2
transcription activity mediated by AKT because MAPK phosphorylation of
the T72 site has been shown to control Ets-2 potency (18, 19). Of note, this MAPK phosphorylation site is conserved in
several members of the Ets family that have been shown to regulate rPRL
promoter activity, including Ets-1, Ets-2, GA binding
protein-
, and Elk-1 (11, 12, 13, 14, 16, 20, 21, 22). While
data from the Murata group clearly implicate an Ets factor as being a
pivotal nuclear effector of PrRP and AKT signaling (7, 15), the identity of the kinase that mediates the direct
phosphorylation of the Ets target protein regulating rPRL promoter
activity remains to be determined. Furthermore, this group has
previously reported that PrRP signals via the MAPK and JNK pathways,
showing that both MAPK and JNK are required for PrRP activation of the
rPRL promoter (15). Taken together with the current report
by this group, PrRP appears to signal via multiple kinase pathways to
the rPRL promoter, including MAPK, JNK, and AKT, and all of these
pathways require an Ets factor. This raises the question as to whether
these three pathways are separate and converging on an Ets factor, or
if some of these kinases are part of a multiprotein complex that
cross-talk. Evidence for the latter is provided by the observation that
AKT and JNK coimmunoprecipitate as a single complex, and that it is
JNK in this complex that phosphorylates Ets-2 (6). While
PrRP can activate these three kinases (MAPK, JNK and AKT),
dominant-negative approaches suggest that disruption of any one of
these kinase pathways is sufficient to completely block PrRP activation
of the rPRL promoter (7, 15). This would imply that these
three kinases are all part of an interconnected pathway, leading
from the PrRP receptor to an Ets factor and the rPRL promoter.
Recent discoveries in the PrRP field have revealed that the PrRP ligand/receptor system may have more widely distributed effects than simply being a putative PRL- releasing factor (23). Nevertheless, most of the key insights on the PrRP biological system have come from its effects on PRL secretion and gene transcription. The paper by Hayakawa et al. (7) continues in this tradition, using the rPRL promoter as a read-out to delineate the signaling pathways mediated by PrRP and has discovered that AKT is an important component of this pathway. However, like all interesting contributions, they often raise many more questions. The questions raised by this paper include: Do the PrRP and AKT responses colocalize to the same cis-acting site? Is this site an Ets binding site? What is the identity of the Ets factor mediating the PrRP/AKT response? Where is the AKT-dependent phosphorylation site(s) on this Ets factor? Which AKT-dependent kinase actually phosphorylates the downstream Ets factor? How is CREB involved in this signaling process? We look forward to the answers to these questions and surely those answers will generate additional interesting questions.
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Footnotes |
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B kinase; JNK, Jun
N-terminal kinase; PrRP, PRL-releasing peptide; rPRL, rat PRL. Received October 25, 2001.
Accepted for publication October 26, 2001.
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References |
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TopIntroductionReferences |
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, and
Pit-1 confer an insulin-responsive phenotype on prolactin promoter
expression in Chinese hamster ovary cells and define the factors
required for insulin-increased transcription. J Biol Chem 276:24931–24936This article has been cited by other articles:
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  G. Vlotides, O. Cooper, Y.-H. Chen, S.-G. Ren, Y. Greenman, and S. Melmed Heregulin Regulates Prolactinoma Gene Expression Cancer Res., May 15, 2009; 69(10): 4209 - 4216. [Abstract] [Full Text] [PDF]
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 T Sakamoto, M Amano, S Hyodo, S Moriyama, A Takahashi, H Kawauchi, and M Ando Expression of prolactin-releasing peptide and prolactin in the euryhaline mudskippers (Periophthalmus modestus): prolactin-releasing peptide as a primary regulator of prolactin J. Mol. Endocrinol., June 1, 2005; 34(3): 825 - 834. [Abstract] [Full Text] [PDF]
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 S. Schinner, A. Barthel, C. Dellas, R. Grzeskowiak, S. K. Sharma, E. Oetjen, R. Blume, and W. Knepel Protein Kinase B Activity Is Sufficient to Mimic the Effect of Insulin on Glucagon Gene Transcription J. Biol. Chem., February 25, 2005; 280(8): 7369 - 7376. [Abstract] [Full Text] [PDF]
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