scholarly journals Identification of calcium-regulated heat-stable protein of 24 kDa (CRHSP24) as a physiological substrate for PKB and RSK using KESTREL

2005 ◽  
Vol 389 (3) ◽  
pp. 775-783 ◽  
Author(s):  
Gillian C. Auld ◽  
David G. Campbell ◽  
Nick Morrice ◽  
Philip Cohen
Keyword(s):  
Growth Factor ◽  
Protein Kinase ◽  
Mammalian Target Of Rapamycin ◽  
Embryonic Stem ◽  
Mitogen Activated Protein Kinase ◽  
Hek 293 ◽  
Human Embryonic Kidney Cells ◽  
Heat Stable ◽  
Heat Stable Protein ◽  
Ribosomal S6 Kinase

A substrate for PKBα (protein kinase Bα) was detected in liver extracts, and was purified and identified as CRHSP24 (calcium-regulated heat-stable protein of apparent molecular mass 24 kDa). PKBα, as well as SGK1 (serum- and glucocorticoid-induced protein kinase 1) and RSK (p90 ribosomal S6 kinase), phosphorylated CRHSP24 stoichiometrically at Ser52in vitro and its brain-specific isoform PIPPin at the equivalent residue (Ser58). CRHSP24 became phosphorylated at Ser52 when HEK-293 (human embryonic kidney) cells were stimulated with IGF-1 (insulin-like growth factor-1) and this was prevented by inhibitors of PI3K (phosphoinositide 3-kinase), but not by rapamycin [an inhibitor of mTOR (mammalian target of rapamycin)] or PD 184352, an inhibitor of the classical MAPK (mitogen-activated protein kinase) cascade and hence the activation of RSK. IGF-1 induced a similar phosphorylation of CRHSP24 in ES (embryonic stem) cells from wild-type mice or mice that express the PDK1 (3-phosphoinositide-dependent kinase 1) mutant (PDK1[L155E]) that activates PKBα normally, but cannot activate SGK. CRHSP24 also became phosphorylated at Ser52 in response to EGF (epidermal growth factor) and this was prevented by blocking activation of both the classical MAPK cascade and the activation of PKBα, but not if just one of these pathways was inhibited. DYRK2 (dual-specificity tyrosine-phosphorylated and -regulated protein kinase 2) phosphorylated CRHSP24 at Ser30, Ser32 and Ser41in vitro, and Ser41 was identified as a site phosphorylated in cells. These and other results demonstrate that CRHSP24 is phosphorylated at Ser52 by PKBα in response to IGF-1, at Ser52 by PKBα and RSK in response to EGF, and at Ser41 in the absence of IGF-1/EGF by a DYRK isoform or another proline-directed protein kinase(s).

scholarly journals Nerve Growth Factor Activates Extracellular Signal-Regulated Kinase and p38 Mitogen-Activated Protein Kinase Pathways To Stimulate CREB Serine 133 Phosphorylation

1998 ◽  
Vol 18 (4) ◽  
pp. 1946-1955 ◽  
Author(s):  
Jun Xing ◽  
Jon M. Kornhauser ◽  
Zhengui Xia ◽  
Elizabeth A. Thiele ◽  
Michael E. Greenberg
Keyword(s):  
Transcription Factor ◽  
Nerve Growth Factor ◽  
Growth Factor ◽  
Protein Kinase ◽  
Mitogen Activated Protein Kinase ◽  
Nerve Growth ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal ◽  
Mitogen Activated Protein ◽  
Ribosomal S6 Kinase

ABSTRACT The mechanisms by which growth factor-induced signals are propagated to the nucleus, leading to the activation of the transcription factor CREB, have been characterized. Nerve growth factor (NGF) was found to activate multiple signaling pathways that mediate the phosphorylation of CREB at the critical regulatory site, serine 133 (Ser-133). NGF activates the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases (MAPKs), which in turn activate the pp90 ribosomal S6 kinase (RSK) family of Ser/Thr kinases, all three members of which were found to catalyze CREB Ser-133 phosphorylation in vitro and in vivo. In addition to the ERK/RSK pathway, we found that NGF activated the p38 MAPK and its downstream effector, MAPK-activated protein kinase 2 (MAPKAP kinase 2), resulting in phosphorylation of CREB at Ser-133. Inhibition of either the ERK/RSK or the p38/MAPKAP kinase 2 pathway only partially blocked NGF-induced CREB Ser-133 phosphorylation, suggesting that either pathway alone is sufficient for coupling the NGF signal to CREB activation. However, inhibition of both the ERK/RSK and the p38/MAPKAP kinase 2 pathways completely abolished NGF-induced CREB Ser-133 phosphorylation. These findings indicate that NGF activates two distinct MAPK pathways, both of which contribute to the phosphorylation of the transcription factor CREB and the activation of immediate-early genes.

scholarly journals Roles for TAB1 in regulating the IL-1-dependent phosphorylation of the TAB3 regulatory subunit and activity of the TAK1 complex

2008 ◽  
Vol 409 (3) ◽  
pp. 711-722 ◽  
Author(s):  
Heidi Mendoza ◽  
David G. Campbell ◽  
Kerry Burness ◽  
James Hastie ◽  
Natalia Ronkina ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Transforming Growth Factor ◽  
Interleukin 1 ◽  
Mitogen Activated Protein Kinase ◽  
Regulatory Subunit ◽  
Hek 293 ◽  
Human Embryonic Kidney Cells ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal ◽  
P38α Mapk

The protein kinase TAK1 (transforming growth factor-β-activated kinase 1), which has been implicated in the activation of MAPK (mitogen-activated protein kinase) cascades and the production of inflammatory mediators by LPS (lipopolysaccharide), IL-1 (interleukin 1) and TNF (tumour necrosis factor), comprises the catalytic subunit complexed to the regulatory subunits, termed TAB (TAK1-binding subunit) 1 and either TAB2 or TAB3. We have previously identified a feedback-control mechanism by which p38α MAPK down-regulates TAK1 and showed that p38α MAPK phosphorylates TAB1 at Ser423 and Thr431. In the present study, we identified two IL-1-stimulated phosphorylation sites on TAB2 (Ser372 and Ser524) and three on TAB3 (Ser60, Thr404 and Ser506) in human IL-1R cells [HEK-293 (human embryonic kidney) cells that stably express the IL-1 receptor] and MEFs (mouse embryonic fibroblasts). Ser372 and Ser524 of TAB2 are not phosphorylated by pathways dependent on p38α/β MAPKs, ERK1/2 (extracellular-signal-regulated kinase 1/2) and JNK1/2 (c-Jun N-terminal kinase 1/2). In contrast, Ser60 and Thr404 of TAB3 appear to be phosphorylated directly by p38α MAPK, whereas Ser506 is phosphorylated by MAPKAP-K2/MAPKAP-K3 (MAPK-activated protein kinase 2 and 3), which are protein kinases activated by p38α MAPK. Studies using TAB1−/− MEFs indicate important roles for TAB1 in recruiting p38α MAPK to the TAK1 complex for the phosphorylation of TAB3 at Ser60 and Thr404 and in inhibiting the dephosphorylation of TAB3 at Ser506. TAB1 is also required to induce TAK1 catalytic activity, since neither IL-1 nor TNFα was able to stimulate detectable TAK1 activity in TAB1−/− MEFs. Surprisingly, the IL-1 and TNFα-stimulated activation of MAPK cascades and IκB (inhibitor of nuclear factor κB) kinases were similar in TAB1−/−, MEKK3−/− [MAPK/ERK (extracellular-signal-regulated kinase) kinase kinase 3] and wild-type MEFs, suggesting that another MAP3K (MAPK kinase kinase) may mediate the IL-1/TNFα-induced activation of these signalling pathways in TAB1−/− and MEKK3−/− MEFs.

scholarly journals Regulation of multisite phosphorylation and 14-3-3 binding of AS160 in response to IGF-1, EGF, PMA and AICAR

2007 ◽  
Vol 407 (2) ◽  
pp. 231-241 ◽  
Author(s):  
Kathryn M. Geraghty ◽  
Shuai Chen ◽  
Jean E. Harthill ◽  
Adel F. Ibrahim ◽  
Rachel Toth ◽  
...  
Keyword(s):  
Growth Factor ◽  
Protein Kinase ◽  
Glucose Transporter ◽  
Kinase Inhibitors ◽  
Hek 293 ◽  
293 Cells ◽  
Ribosomal S6 Kinase ◽  
Phosphoinositide 3 Kinase ◽  
Amp Activated Protein Kinase

AS160 (Akt substrate of 160 kDa) mediates insulin-stimulated GLUT4 (glucose transporter 4) translocation, but is widely expressed in insulin-insensitive tissues lacking GLUT4. Having isolated AS160 by 14-3-3-affinity chromatography, we found that binding of AS160 to 14-3-3 isoforms in HEK (human embryonic kidney)-293 cells was induced by IGF-1 (insulin-like growth factor-1), EGF (epidermal growth factor), PMA and, to a lesser extent, AICAR (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside). AS160-14-3-3 interactions were stabilized by chemical cross-linking and abolished by dephosphorylation. Eight residues on AS160 (Ser318, Ser341, Thr568, Ser570, Ser588, Thr642, Ser666 and Ser751) were differentially phosphorylated in response to IGF-1, EGF, PMA and AICAR. The binding of 14-3-3 proteins to HA–AS160 (where HA is haemagglutinin) was markedly decreased by mutation of Thr642 and abolished in a Thr642Ala/Ser341Ala double mutant. The AGC (protein kinase A/protein kinase G/protein kinase C-family) kinases RSK1 (p90 ribosomal S6 kinase 1), SGK1 (serum- and glucocorticoid-induced protein kinase 1) and PKB (protein kinase B) displayed distinct signatures of AS160 phosphorylation in vitro: all three kinases phosphorylated Ser318, Ser588 and Thr642; RSK1 also phosphorylated Ser341, Ser751 and to a lesser extent Thr568; and SGK1 phosphorylated Thr568 and Ser751. AMPK (AMP-activated protein kinase) preferentially phosphorylated Ser588, with less phosphorylation of other sites. In cells, the IGF-1-stimulated phosphorylations, and certain EGF-stimulated phosphorylations, were inhibited by PI3K (phosphoinositide 3-kinase) inhibitors, whereas the RSK inhibitor BI-D1870 inhibited the PMA-induced phosphorylations. The expression of LKB1 in HeLa cells and the use of AICAR in HEK-293 cells promoted phosphorylation of Ser588, but only weak Ser341 and Thr642 phosphorylations and binding to 14-3-3s. Paradoxically however, phenformin activated AMPK without promoting AS160 phosphorylation. The IGF-1-induced phosphorylation of the novel phosphorylated Ser666-Pro site was suppressed by AICAR, and by combined mutation of a TOS (mTOR signalling)-like sequence (FEMDI) and rapamycin. Thus, although AS160 is a common target of insulin, IGF-1, EGF, PMA and AICAR, these stimuli induce distinctive patterns of phosphorylation and 14-3-3 binding, mediated by at least four protein kinases.

scholarly journals Disruption of 3-Phosphoinositide-dependent Kinase 1 (PDK1) Signaling by the Anti-tumorigenic and Anti-proliferative AgentN-α-tosyl-l-phenylalanyl Chloromethyl Ketone

2001 ◽  
Vol 276 (15) ◽  
pp. 12466-12475 ◽  
Author(s):  
Bryan A. Ballif ◽  
Akiko Shimamura ◽  
Eunice Pae ◽  
John Blenis
Keyword(s):  
Protein Kinase ◽  
Biological Effects ◽  
Embryonic Stem ◽  
Mitogen Activated Protein Kinase ◽  
S6 Kinase ◽  
Chloromethyl Ketone ◽  
Cellular Targets ◽  
Mitogen Activated Protein ◽  
Ribosomal S6 Kinase ◽  
Phosphoinositide Dependent Kinase

The anti-tumorigenic and anti-proliferative effects ofN-α-tosyl-l-phenylalanyl chloromethyl ketone (TPCK) have been known for more than three decades. Yet little is known about the discrete cellular targets of TPCK controlling these effects. Previous work from our laboratory showed TPCK, like the immunosuppressant rapamycin, to be a potent inhibitor of the 70-kilodalton ribosomal S6 kinase 1 (S6K1), which mediates events involved in cell growth and proliferation. We show here that rapamycin and TPCK display distinct inhibitory mechanisms on S6K1 as a rapamycin-resistant form of S6K1 was TPCK-sensitive. Additionally, we show that TPCK inhibited the activation of the related kinase and proto-oncogene Akt. Upstream regulators of S6K1 and Akt include phosphoinositide 3-kinase (PI 3-K) and 3-phosphoinositide-dependent kinase 1 (PDK1). Whereas TPCK had no effect on either mitogen-regulated PI 3-K activity or total cellular PDK1 activity, TPCK prevented phosphorylation of the PDK1 regulatory sites in S6K1 and Akt. Furthermore, whereas both PDK1 and the mitogen-activated protein kinase (MAPK) are required for full activation of the 90-kilodalton ribosomal S6 kinase (RSK), TPCK inhibited RSK activation without inhibiting MAPK activation. Consistent with the capacity of RSK and Akt to mediate a cell survival signal, in part through phosphorylation of the pro-apoptotic protein BAD, TPCK reduced BAD phosphorylation and led to cell death in interleukin-3-dependent 32D cells. Finally, in agreement with results seen in embryonic stem cells lacking PDK1, protein kinase A activation was not inhibited by TPCK showing TPCK specificity for mitogen-regulated PDK1 signaling. TPCK inhibition of PDK1 signaling thus disables central kinase cascades governing diverse cellular processes including proliferation and survival and provides an explanation for its striking biological effects.

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