Biomedical subjects

John Blenis

Publications and source records attributed to John Blenis.

At least 19 recordsLinked to original sources

PubMed2024

Genome-wide CRISPR screens in spheroid culture reveal that the tumor suppressor LKB1 inhibits growth via the PIKFYVE lipid kinase.

The tumor suppressor LKB1 is a serine/threonine protein kinase that is frequently mutated in human lung adenocarcinoma (LUAD). LKB1 regulates a complex signaling network that is known to control cell polarity and metabolism; however, the pathways that mediate the tumor-suppressive activity of LKB1 are incompletely defined. To identify mechanisms of LKB1-mediated growth suppression, we developed a spheroid-based cell culture assay to study LKB1-dependent growth. We then performed genome-wide CRISPR screens in spheroidal culture and found that LKB1 suppresses growth, in part, by activating the PIKFYVE lipid kinase. Finally, we used chemical inhibitors and a pH-sensitive reporter to determine that LKB1 impairs growth by promoting the internalization of wild-type EGFR in a PIKFYVE-dependent manner.

Humans↗

PubMed2007

SHP-2 regulates cell growth by controlling the mTOR/S6 kinase 1 pathway.

Cell growth (accumulation in cell mass) ensues through the promotion of macromolecular biosynthesis. S 6 ribosomal kinase 1 (S6K1), which is activated by the mammalian target of rapamycin, is critical for cell growth. The early events that control S6K1 signaling remain unclear. Here we show that SHP-2 suppresses S6K1 activity under conditions of growth factor deprivation. We show that under conditions of growth factor deprivation, S6K1 activity was increased in fibroblasts lacking functional SHP-2 and in cells where knock down of SHP-2 expression was established by small interference RNA. Consistent with these findings, fibroblasts lacking functional SHP-2 exhibited increased cell size as compared with wild type cells. Growth factor deprivation reduces cellular energy, and the energy-sensing 5'-AMP-activated protein kinase (AMPK) negatively regulates S6K1. We found that SHP-2 promoted AMPK activity under conditions of growth factor deprivation (low energy), suggesting that SHP-2 negatively regulates S6K1 via an AMPK-dependent pathway. These results implicate SHP-2 as an early mediator in the S6K1 signaling pathway to limit cell growth in low energy states.

Adenylate Kinase↗

PubMed2006

Mind the GAP: Wnt steps onto the mTORC1 train.

The TSC1/2 tumor-suppressor complex controls protein synthesis through the regulation of mTOR. In this issue of Cell, Inoki et al. (2006) report that the kinases GSK3 and AMPK cooperate in the activation of TSC2 to inhibit mTOR activity. Surprisingly, the phosphorylation of TSC2 by GSK3 is markedly suppressed by Wnt signaling. This suggests that components of the mTOR pathway may be therapeutic targets for diseases linked to hyperactive Wnt signaling.

AMP-Activated Protein Kinases↗

PubMed2006

The mTOR/PI3K and MAPK pathways converge on eIF4B to control its phosphorylation and activity.

The eukaryotic translation initiation factor 4B (eIF4B) plays a critical role in recruiting the 40S ribosomal subunit to the mRNA. In response to insulin, eIF4B is phosphorylated on Ser422 by S6K in a rapamycin-sensitive manner. Here we demonstrate that the p90 ribosomal protein S6 kinase (RSK) phosphorylates eIF4B on the same residue. The relative contribution of the RSK and S6K modules to the phosphorylation of eIF4B is growth factor-dependent, and the two phosphorylation events exhibit very different kinetics. The S6K and RSK proteins are members of the AGC protein kinase family, and require PDK1 phosphorylation for activation. Consistent with this requirement, phosphorylation of eIF4B Ser422 is abrogated in PDK1 null embryonic stem cells. Phosphorylation of eIF4B on Ser422 by RSK and S6K is physiologically significant, as it increases the interaction of eIF4B with the eukaryotic translation initiation factor 3.

3-Phosphoinositide-Dependent Protein Kinases↗

PubMed2006

Inhibition of ERK-MAP kinase signaling by RSK during Drosophila development.

Although p90 ribosomal S6 kinase (RSK) is known as an important downstream effector of the ribosomal protein S6 kinase/extracellular signal-regulated kinase (Ras/ERK) pathway, its endogenous role, and precise molecular function remain unclear. Using gain-of-function and null mutants of RSK, its physiological role was successfully characterized in Drosophila. Surprisingly, RSK-null mutants were viable, but exhibited developmental abnormalities related to an enhanced ERK-dependent cellular differentiation such as ectopic photoreceptor- and vein-cell formation. Conversely, overexpression of RSK dramatically suppressed the ERK-dependent differentiation, which was further augmented by mutations in the Ras/ERK pathway. Consistent with these physiological phenotypes, RSK negatively regulated ERK-mediated developmental processes and gene expressions by blocking the nuclear localization of ERK in a kinase activity-independent manner. In addition, we further demonstrated that the RSK-dependent inhibition of ERK nuclear migration is mediated by the physical association between ERK and RSK. Collectively, our study reveals a novel regulatory mechanism of the Ras/ERK pathway by RSK, which negatively regulates ERK activity by acting as a cytoplasmic anchor in Drosophila.

Active Transport, Cell Nucleus↗

PubMed2006

MAPK signal specificity: the right place at the right time.

Although the mechanisms that lead to activation of the Ras, extracellular-signal-regulated kinase mitogen-activated protein kinase (Ras/ERK-MAPK) signaling pathway have been studied intensively, the fundamental principles that determine how activation of ERK signaling can result in distinct biological outcomes have only recently received attention. Factors such as cell-surface receptor density, expression of scaffolding proteins, the surrounding extracellular matrix, and the interplay between kinases and phosphatases modulate the strength and duration of ERK signaling. Furthermore, the spatial distribution and temporal qualities of ERK can markedly alter the qualitative and quantitative features of downstream signaling to immediate early genes (IEG) and the expression of IEG-encoded protein products. As a result, IEG products provide a molecular interpretation of ERK dynamics, enabling the cell to program an appropriate biological response.

Animals↗

PubMed2006

A specific mechanomodulatory role for p38 MAPK in embryonic joint articular surface cell MEK-ERK pathway regulation.

Mechanisms regulating cell behavior and extracellular matrix composition in response to mechanical stimuli remain unresolved. Our previous studies have established that the MEK-ERK cascade plays a specific role in the mechano-dependent joint formation process by promoting the assembly of pericellular matrices reliant upon hyaluronan (HA) for their integrity. Here we demonstrate: (i) novel cross-talk between p38 MAPK and MEK-ERK signaling pathways that is specific for mechanical stimuli and (ii) a role for p38 MAPK in facilitating HA production by cells derived from the articular surface of embryonic chick tibiotarsal joints. We find that p38 MAPK blockade restricts pericellular assembly of HA-rich matrices and reduces basal as well as mechanical strain-induced release of HA. p38 MAPK blockers potentiated early strain-induced increases but restricted sustained increases in MEK/ERK phosphorylation at later times; c-Fos hyperphosphorylation at threonine 325 was found to parallel this p38 MAPK-mediated modulation of ERK activation. In contrast, p38 MAPK inhibitors had no detectable effect on the ERK activation induced by fibroblast growth factor 2 or pervanadate, a phosphatase inhibitor, and MEK inhibitors did not influence p38 MAPK phosphorylation, confirming both the specificity and unidirectionality of p38 MAPK-ERK cross-talk. Immunochemical and immunoblotting studies revealed constitutive p38 MAPK activation in cells at, or derived from, developing articular joint surfaces. Unlike the MEK-ERK pathway, however, p38 MAPK was not further stimulated by mechanical stimulation in vitro. Thus, p38 MAPK specifically facilitates ERK activation and downstream signaling in response to mechanical stimuli. These results suggest that constitutively active p38 MAPK serves an essential, permissive role in mechanically induced changes in ERK activation and in the accumulation of HA-rich extracellular matrices that serve a key role in joint development.

Animals↗

PubMed2006

TORgeting oncogene addiction for cancer therapy.

The PI3K-Akt-mTOR growth-regulating pathway is conserved from mammals to flies and hyperactivated in many cancers. Accordingly, rapamycin analogs, which are inhibitors of mTOR-Raptor signaling, have recently garnered much interest as potential therapeutic agents against cancer. However, due to the heterogeneity of tumors, prior knowledge of the genetic and biochemical background of cancer cells will be required for effective targeted therapy. Thus, the identification of biological markers against activated oncogenic pathways is needed. In the January issue of Nature Medicine, Thomas et al. identify the loss of VHL tumor suppressor gene as a potential determining factor in tumor sensitivity to rapamycin.

Animals↗

PubMed2006

Rheb activation of mTOR and S6K1 signaling.

More than 10 years ago, Rheb (Ras homolog enriched in brain) was identified as a highly conserved protein that is a member of the Ras superfamily of small GTPases, which play critical roles in cell growth and proliferation. Recently, a convergence of genetic and biochemical evidence from yeast, Drosophila, and mammalian cells has placed Rheb upstream of the mammalian target of rapamycin (mTOR) and immediately downstream of the tumor suppressors TSC1 (hamartin) and TSC2 (tuberin). Rheb plays a key role in the regulation of cell growth in response to growth factors, nutrients, and amino acids linking PI3K and TOR signaling. Rheb activation of the nutrient and energy-sensitive TOR pathway leads to the direct phosphorylation of two known downstream translational control targets by mTOR, the 40S ribosomal S6 kinase 1 (S6K1) and the eukaryotic translation initiation factor 4E (eIF4E)- binding protein 1 (4E-BP1). Appropriate regulation of this pathway is crucial for the proper control of cell growth, proliferation, survival, and differentiation. Inappropriate regulation of these signaling molecules, therefore, can lead to a variety of human diseases. In this chapter, we describe cell biological and biochemical methods commonly used to study Rheb activation and dissect its role in the mTOR-signaling pathway.

Animals↗

PubMed2005

mTOR and S6K1 mediate assembly of the translation preinitiation complex through dynamic protein interchange and ordered phosphorylation events.

In response to nutrients, energy sufficiency, hormones, and mitogenic agents, S6K1 phosphorylates several targets linked to translation. However, the molecular mechanisms whereby S6K1 is activated, encounters substrate, and contributes to translation initiation are poorly understood. We show that mTOR and S6K1 maneuver on and off the eukaryotic initiation factor 3 (eIF3) translation initiation complex in a signal-dependent, choreographed fashion. When inactive, S6K1 associates with the eIF3 complex, while the S6K1 activator mTOR/raptor does not. Cell stimulation promotes mTOR/raptor binding to the eIF3 complex and phosphorylation of S6K1 at its hydrophobic motif. Phosphorylation results in S6K1 dissociation, activation, and subsequent phosphorylation of its translational targets, including eIF4B, which is then recruited into the complex in a phosphorylation-dependent manner. Thus, the eIF3 preinitiation complex acts as a scaffold to coordinate a dynamic sequence of events in response to stimuli that promote efficient protein synthesis.

Adaptor Proteins, Signal Transducing↗

PubMed2005

The tumor suppressor DAP kinase is a target of RSK-mediated survival signaling.

The viability of vertebrate cells depends on a complex signaling interplay between survival factors and cell-death effectors. Subtle changes in the equilibrium between these regulators can result in abnormal cell proliferation or cell death, leading to various pathological manifestations. Death-associated protein kinase (DAPK) is a multidomain calcium/calmodulin (CaM)-dependent Ser/Thr protein kinase with an important role in apoptosis regulation and tumor suppression. The molecular signaling mechanisms regulating this kinase, however, remain unclear. Here, we show that DAPK is phosphorylated upon activation of the Ras-extracellular signal-regulated kinase (ERK) pathway. This correlates with the suppression of the apoptotic activity of DAPK. We demonstrate that DAPK is a novel target of p90 ribosomal S6 kinases (RSK) 1 and 2, downstream effectors of ERK1/2. Using mass spectrometry, we identified Ser-289 as a novel phosphorylation site in DAPK, which is regulated by RSK. Mutation of Ser-289 to alanine results in a DAPK mutant with enhanced apoptotic activity, whereas the phosphomimetic mutation (Ser289Glu) attenuates its apoptotic activity. Our results suggest that RSK-mediated phosphorylation of DAPK is a unique mechanism for suppressing the proapoptotic function of this death kinase in healthy cells as well as Ras/Raf-transformed cells.

Animals↗

PubMed2005

Analysis of mTOR signaling by the small G-proteins, Rheb and RhebL1.

The small G protein Rheb (Ras homologue enriched in brain) is known to promote mammalian target of rapamycin (mTOR) signaling. In this study, we show that Rheb like-1 protein (RhebL1) rescues mTOR signaling during nutrient withdrawal and that tuberous sclerosis complex-1 (TSC) and TSC2 impairs RhebL1-mediated signaling through mTOR. We identify critical residues within the switch I region (N41) and 'constitutive' effector (Ec) region (Y/F54 and L56) of Rheb and RhebL1, which are required for their efficient activation of mTOR signaling. Mutation of Rheb and RhebL1 at N41 impaired their interaction with mTOR, which identifies mTOR as a common downstream target of both Rheb and RhebL1.

Amino Acid Sequence↗

PubMed2005

Spatially separate docking sites on ERK2 regulate distinct signaling events in vivo.

Inhibitors of the oncogenic Ras-MAPK pathway have been intensely pursued as therapeutics. Targeting this pathway, however, presents challenges due to the essential role of MAPK in homeostatic functions. The phosphorylation and activation of MAPK substrates is regulated by protein-protein interactions with MAPK docking sites. Active ERK1/2 (extracellular signal-regulated kinase 1/2)-MAPKs localize to effectors containing DEF (docking site for ERK, (F)/(Y) -X-(F)/(Y) -P)- or D-domain (docking domain) motifs. We have examined the in vivo activity of ERK2 mutants with impaired ability to signal via either docking site. Mutations in the DEF-domain binding pocket prevent activation of DEF-domain-containing effectors but not RSK (90 kDa ribosomal S6 kinase), which contains a D domain. Conversely, mutation of the ERK2 CD domain, which interacts with D domains, prevents RSK activation but not DEF-domain signaling. Uncoupling docking interactions does not compromise ERK2 phosphotransferase activity. ERK2 DEF mutants undergo regulated nuclear translocation but are defective for Elk-1/TCF transactivation and target gene induction. Thus, downstream branches of ERK2 signaling can be selectively inhibited without blocking total pathway activity. Significantly, several protooncogenes contain DEF domains and are regulated by ERK1/2. Therefore, disrupting ERK-DEF domain interactions could be an alternative to inhibiting oncogenic Ras-MAPK signaling.

Animals↗

PubMed2005

Identification of S6 kinase 1 as a novel mammalian target of rapamycin (mTOR)-phosphorylating kinase.

Here we demonstrate that mammalian target of rapamycin (mTOR) is phosphorylated in a rapamycin-sensitive manner. We show that S6 kinase 1 (S6K1), but not Akt, directly phosphorylates mTOR in cell-free in vitro system and in cells. Expression of a constitutively active, rapamycin- and wortmannin-resistant S6K1 leads to constitutive phosphorylation of mTOR, whereas knock-down of S6K1 using small inhibitory RNA greatly reduces mTOR phosphorylation despite elevated Akt activity. Importantly, phosphorylation of mTOR by S6K1 occurs at threonine 2446/serine 2448. This region has been shown previously to be part of a regulatory repressor domain. These sites are also constitutively phosphorylated in the breast cancer cell line MCF7 carrying an amplification of the S6K1 gene, but not in a less tumorigenic cell line, MCF10a. Many models for Akt signaling to mTOR have been presented, suggesting direct phosphorylation by Akt. These models must be reconsidered in light of the present findings.

Alleles↗

PubMed2005

Sensitized RNAi screen of human kinases and phosphatases identifies new regulators of apoptosis and chemoresistance.

Evasion from apoptosis is a hallmark of cancer, and recent success using targeted therapeutics underscores the importance of identifying anti-apoptotic survival pathways. Here we utilize RNA interference (RNAi) to systematically screen the kinase and phosphatase component of the human genome. In addition to known kinases, we identified several new survival kinases. Interestingly, numerous phosphatases and associated regulatory subunits contribute to cell survival, revealing a previously unrecognized general role for phosphatases as negative regulators of apoptosis. We also identified a subset of phosphatases with tumour-suppressor-like activity. Finally, RNAi targeting of specific protein kinases sensitizes resistant cells to chemotherapeutic agents. The development of inhibitors that target these kinases or phosphatases may lead to new anti-cancer strategies.

Apoptosis↗

PubMed2005

Characterization of a conserved C-terminal motif (RSPRR) in ribosomal protein S6 kinase 1 required for its mammalian target of rapamycin-dependent regulation.

The mammalian target of rapamycin, mTOR, is a Ser/Thr kinase that promotes cell growth and proliferation by activating ribosomal protein S6 kinase 1 (S6K1). We previously identified a conserved TOR signaling (TOS) motif in the N terminus of S6K1 that is required for its mTOR-dependent activation. Furthermore, our data suggested that the TOS motif suppresses an inhibitory function associated with the C terminus of S6K1. Here, we have characterized the mTOR-regulated inhibitory region within the C terminus. We have identified a conserved C-terminal "RSPRR" sequence that is responsible for an mTOR-dependent suppression of S6K1 activation. Deletion or mutations within this RSPRR motif partially rescue the kinase activity of the S6K1 TOS motif mutant (S6K1-F5A), and this rescued activity is rapamycin resistant. Furthermore, we have shown that the RSPRR motif significantly suppresses S6K1 phosphorylation at two phosphorylation sites (Thr-389 and Thr-229) that are crucial for S6K1 activation. Importantly, introducing both the Thr-389 phosphomimetic and RSPRR motif mutations into the catalytically inactive S6K1 mutant S6K1-F5A completely rescues its activity and renders it fully rapamycin resistant. These data show that the N-terminal TOS motif suppresses an inhibitory function mediated by the C-terminal RSPRR motif. We propose that the RSPRR motif interacts with a negative regulator of S6K1 that is normally suppressed by mTOR.

Amino Acid Motifs↗

PubMed2005

Quantitative phosphorylation profiling of the ERK/p90 ribosomal S6 kinase-signaling cassette and its targets, the tuberous sclerosis tumor suppressors.

Reversible protein phosphorylation is an essential cellular regulatory mechanism. Many proteins integrate and are modulated by multiple phosphorylation events derived from complex signaling cues. Simultaneous detection and quantification of temporal changes in all of a protein's phosphorylation sites could provide not only an immediate assessment of a known biochemical activity but also important insights into molecular signaling mechanisms. Here we show the use of stable isotope-based quantitative MS to globally monitor the kinetics of complex, ordered phosphorylation events on protein players in the canonical mitogen-activated protein kinase signaling pathway. In excellent agreement with activity assays and phosphospecific immunoblotting with the same samples, we quantified epidermal growth factor-induced changes in nine phosphorylation sites in the extracellular signal-regulated kinase (ERK)/p90 ribosomal S6 kinase-signaling cassette. Additionally, we monitored 14 previously uncharacterized and six known phosphorylation events after phorbol ester stimulation in the ERK/p90 ribosomal S6 kinase-signaling targets, the tuberous sclerosis complex (TSC) tumor suppressors TSC1 and TSC2. By using quantitative phosphorylation profiling in conjunction with pharmacological kinase inhibitors we uncovered a ERK-independent, protein kinase C-dependent pathway to TSC2 phosphorylation. These results establish quantitative phosphorylation profiling as a means to simultaneously identify, quantify, and delineate the kinetic changes of ordered phosphorylation events on a given protein and defines parameters for the rapid discovery of important in vivo phosphoregulatory mechanisms.

Binding Sites↗

PubMed2005

Graded mitogen-activated protein kinase activity precedes switch-like c-Fos induction in mammalian cells.

The mitogen-activated protein kinase (MAPK) pathway is an evolutionarily conserved signaling module that controls important cell fate decisions in a variety of physiological contexts. During Xenopus oocyte maturation, the MAPK cascade converts an increasing progesterone stimulus into a switch-like, all-or-nothing response. While the importance of such switch-like behavior is widely discussed in the literature, it is not known whether the MAPK pathway in mammalian cells exhibits a switch-like or graded response. For this study, we used flow cytometry and immunofluorescence to generate single-cell measurements of MAPK signaling in Swiss 3T3 fibroblasts. In contrast to the case in Xenopus oocytes, we found that ERK activation in individual mammalian cells is not ultrasensitive and shows a graded response to changes in agonist concentration. Thus, the conserved MAPK signaling module exhibits different systems-level properties in different cellular contexts. Furthermore, the graded ERK response was converted into a more switch-like behavior at the level of immediate-early gene induction and cell cycle progression. Thus, while MAPK signaling is involved in all-or-nothing cell fate decisions for both Xenopus oocyte maturation and mammalian fibroblast proliferation, the underlying mechanisms responsible for the switch-like nature of the cellular responses are different in these two systems, with the mechanism appearing to lie downstream of the kinase cascade in mammalian fibroblasts.

Animals↗