Each week we showcase a hot new cell signaling article from a Nature Publishing Group journal. Free full text access to the paper will be maintained for three months, after which the paper will be accessible via the Research Library.
Protein-protein interaction networks: To party or date?
Protein-protein interaction networks, or 'interactomes', provide a systems level view of cellular processes like signal transduction, although they have so far been largely considered as static networks. Within these networks, most proteins interact with few partners. However, a small number of proteins - called 'hubs' - interact with many different partners. Scale-free interactome networks are particularly vulnerable to the removal of hubs - knocking out yeast hub genes results in a threefold increase in lethality, compared with the removal of peripheral proteins. Han et al. have analyzed the properties of these hubs taking temporal and spatial dynamics into account. They conclude that hubs fall into one of two categories: 'party hubs', which interact with most of their partners simultaneously, and 'date hubs', which bind different partners at different locations and times.
The biological role of topological hubs may vary depending upon the timing and location of the interactions they mediate. The authors used a filtered yeast interactome (FYI), compiled from different sets of yeast mRNA expression profiling data. The FYI contained 1,379 proteins with an average of 3.6 interactions per protein. The average Pearson's correlation coefficient (PCC) was used to measure the strength of the linear relationship between each hub and its partners. A bimodal pattern of distribution was most clearly apparent for two conditions analyzed, the 'stress response' and 'cell cycle', indicating two distinct hub populations: 'party hubs' with a high average PCC and 'date hubs' with a low PCC.
'Date hub' partners are significantly more diverse in subcellular location than 'party hub' partners. Upon the removal of 'party hubs', general network connectivity was unaffected. However, 'date hub' removal resembled the effects seen by deletion of all hubs. The network remaining after the removal of 'party hubs' is significantly greater than that left behind after the removal of date hubs. In fact, the FYI subnetworks removed with 'date hubs' correspond to small modules of specific biological processes. These sub-networks represent not only stable molecular machinery but also loosely connected regulatory pathways such as osmosensing.
Thus, a model emerges for organized modularity where 'date hubs' represent global connectors between modules, and 'party hubs' function inside modules. This suggests that 'date hubs' have a central role in the modular organization of the yeast proteome. For example, the 'date hub' calmodulin connects four different biological modules, 'cation homeostasis', 'protein folding and stabilization', 'budding, cell polarity and filament formation' and 'endoplasmic reticulum', whereas the party hubs Sec17, Sec22, and Vti1 all function within the 'endoplasmic reticulum' module. The proteome is more sensitive to perturbation of date hubs than that of party hubs.
This spatio-temporal dynamic analysis may be extended to other systems such as the World Wide Web, social and epidemiological networks.
Brenda Riley, Assistant Editor Signaling Gateway
article
Jing-Dong J. Han, Nicolas Bertin, Tong Hao, Debra S. Goldberg, Gabriel F. Berriz, Lan V. Zhang, Denis Dupuy, Albertha J. M. Walhout, Michael E. Cusick, Frederocl P. Roth & Marc Vidal Evidence for dynamically organized modularity in the yeast protein-protein interaction network Nature, 430, 88 - 93 (01 July 2004); doi:10.1038/nature02555
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Protein phosphatase 1 structure: How MYPT1 adds specificity
The reciprocal regulation of a plethora of substrates by Serine/threonine (Ser/Thr) protein kinases and phosphatases is at the heart of signal transduction. This well-tuned balance of transient phosphorylation occurs even though Ser-Thr kinases significantly outnumber phosphatases. The ubiquitous protein phosphatase 1 (PP1) is an effective catalyst but lacks inherent substrate specificity, which is brought about by interaction with a spectrum of regulatory subunits that only share a canonical PP1-binding sequence known as the RVxF motif. Terrak et al. now show that structural elements of the myosin phosphatase targeting subunit (MYPT1) reshape the catalytic cleft of PP1, thereby increasing PP1's catalytic activity and specificity for myosin.
Smooth muscle relaxation occurs through the dephosphorylation of myosin. Myosin phosphatase is made up of a trimeric holoenzyme consisting of MYPT1, PP1 and M20, a protein of unknown function. Binding of PP1 to the N-terminal region of MYPT1 produces a 15-fold increase in catalytic activity and a 10-fold higher affinity for phopshorylated myosin. The authors report the 2.7-Å-resolution structure of the PP1δ-MYPT1 complex.
The overall structure of PP1 consists of two tightly linked domains, an N-terminal α/β domain, and a C-terminal β domain that also contains three α-helices. The β-strands converge to a β-sandwich at the inter-domain interface. At this interface the catalytic site resides within a large Y-shaped cleft formed by three converging grooves (the hydrophobic, acidic and C-terminal grooves). Upon binding, the 34 N-terminal amino acids of MYPT1 proximal to the RVxF motif, form a long arm that wraps around PP1 to reach the base of the Y-shaped catalytic cleft.
Amino acids 39-291 of MYPT1 fold into two groups of four ankyrin repeats separated by a hinge at Glu 172. MYPT1 interacts with PP1 via repeats 1, 5, 6, and 7, with repeat 1 interacting with the RVxF-binding pocket. Not only do the ankyrin repeats play a role in binding, but they also play a key role in the modulation of PP1 catalytic activity: β-hairpin loops from the two groups of ankyrin repeats face each other forming a clamp-like structure that closes around the C-terminus of PP1, and this gives rise to a large acidic cleft which is positioned so as to extend the catalytic cleft of PP1. This extended catalytic cleft is specifically adapted for the myosin substrate and is less compatible with other substrates. In this way, the regulatory subunit adapts the catalytic cleft of PP1 to perform a specific function. These features of the PP1-MYPT1 complex have general implications for how other PP1 regulatory subunits work.
Brenda Riley, Assistant Editor Signaling Gateway
article
Mohammed Terrak, Frederic Kerff, Knut Langsetmo, Terence Tao & Roberto Dominguez Structural basis of protein phosphatase 1 regulation Nature, 429, 780 - 784, (17 June 2004); doi:10.1038/nature02582
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PI3K/Akt signaling: TORmenting prostate cancer
PTEN
is a tumor suppressor gene that is frequently deleted or mutated
in prostate, endometrial and breast cancer. This lipid phosphatase
regulates cell survival, growth and migration by suppressing phosphoinositol
3 kinase (PI3K) signaling, thereby down-regulating downstream
mediators, including the oncogenic serine-threonine kinase AKT,
which in turn regulates the mTOR
(mammalian target of rapamycin) growth pathway. Clinical trials
are currently underway to evaluate mTOR inhibition as a viable
cancer treatment for PTEN-null cancers. Majumder et al.
now show that the anti-tumor response to mTOR inhibition occurs
through independent apoptotic and Hif-1α
regulatory pathways.
The authors previously found that transgenic mice expressing
activated AKT1
in the epithelial cells of the ventral prostate results in a phenotype
similar to prostatic intraepithelial neoplasia (PIN). These AKT1-Tg
mice showed no signs of PIN after two weeks of treatment with
the rapamycin derivative RAD001. RAD001 selectively inhibits mTOR
activity downstream of AKT, without altering other elements of
Akt signaling; thus, the AKT-dependent PIN phenotype requires
mTOR activity.
While investigating the time course of the response to RAD001,
the authors noted a lack of proliferating cells, as well as rapid
phenotypic reversion and a vacuolated cell appearance. This suggested
that the loss of intraluminal cells after RAD001 exposure may
be due to apoptosis and that the intraluminal epithelial cells
in PIN depend on an mTOR-dependent, antiapoptotic signaling pathway
for their survival. Indeed, when AKT1-Tg mice were crossed
with transgenic mice expressing BCL2
in the ventral prostate, the progeny exhibited only partial regression
of the phenotype when treated with RAD001. This reveals that BCL2
expression blocks the induction of apoptosis by mTOR inhibition
and leads to a partial resistance to RAD001. Thus, apoptosis in
response to mTOR inhibition requires the mitochondrial apoptotic
pathway.
Gene expression profiling was carried out to determine if another pathway is involved in RAD001 inhibition of PIN. This sceen identified a set of hypoxia-inducible factor 1 (HIF-1) target genes that were induced in the transgenic mice, and repressed by RAD001. Indeed, HIF-1α activity was found to be upregulated in both AKT and AKT/BCL2-expressing tumors, and downregulated after RAD001 administration. These changes preceeded any phenotypic changes and are likely to be direct, thus pointing to a functional role for HIF-1α in this pathway.
Together, these results define two mechanisms likely to be instrumental in the anti-oncogenic response of mTOR inhibition. Prostate cancer patients with overexpression of BCL2 in tumors, or TOR-independent activation of HIF-1α, may be less responsive to mTOR inhibition treatment. This may allow tailoring of prostatic cancer treatments to patients.
Brenda Riley, Assistant Editor Signaling Gateway
article
Pradip K Majumder, Phillip G Febbo, Rachel Bikoff, Raanan Berger, Qi Xue, Louis M McMahon, Judith Manola, James Brugarolas, Timothy J McDonnell, Todd R Golub, Massimo Loda, Heidi A Lane & William R Sellers mTOR inhibition reverses Akt-dependent prostate intraepithelial neoplasia through regulation of apoptotic and HIF-1-dependent pathways Nature Medicine , 10, 594 - 601, (2004); doi:10.1038/nm1052
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news and views:
Ingo K Mellinghoff & Charles L Sawyers TORward AKTually useful mouse models Nature Medicine , 10, 579 - 580 (2004); doi:10.1038/nm0604-579
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Cardiac hypertrophy: Stretching to your heart's content
Cardiac hypertrophy is an adaptational state of increased heart muscle mass in response to increased cardiac load. It is a well established risk factor for cardiac failure. While mechanical stress is the most important stimulus for cardiac hypertrophy, vasoactive peptides, catecholamines, cytokines and growth factors also contribute.
It is well established that inhibition of the angiotension II (AII) type 1 (AT1) receptor prevents progression towards heart failure through the attenuation of cardiac hypertrophy. It has been reported that AII stored in cardiomyocytes is released into culture medium upon mechanical stress, resulting in the autocrine induction of cardiomyocyte hypertrophy. Zou et al. have now developed an in vitro device whereby stretch stimuli can be imposed on cultured cardiomyocytes, revealing that mechanical stress can directly activate the AT1 receptor independently of AII.
The authors confirmed that the AT1 receptor is involved in the activation of extracellular-signal-related kinases (ERKs) after mechanical stress. However, contrary to previous research, mechanical stress-induced secretion of AII is insufficient to fully activate the AT1 receptor and does not play a significant role in ERK activation. Upon mechanical stress, ERK activation occurred in cells expressing an AT1 receptor with a mutated ligand binding site as well as in AII null cardiomyocytes. This was confirmed in vivo, indicating that mechanical stress is able to activate ERKs through the AT1 receptor even in the absence of AII.
The AT1 receptor belongs to the G protein-coupled receptor family. ERK activation was weaker in cells expressing G protein binding deficient AP1 receptor mutants, suggesting that AP-1 receptor-G protein coupling is partly involved in ERK activation. On the other hand, mechanical stress induced an association between the non-receptor tyrosine kinase Janus kinase 2 (Jak2) and the AT1 receptor. It also increased Jak2 phosphorylation and ERK activation independently of G protein coupling. Overexpression of the AT1 receptor resulted in a fivefold increase in levels of inositol phosphates, which was further increased by mechanical stretch.
Together, these results suggest that the AT1 receptor acts as a 'mechanical sensor', converting mechanical stress into biochemical signals inside the cell without the involvement of AII. Although the mechanism remains ill defined, the authors propose that stretching the cell membrane may either directly change the conformation of the AT1 receptor, or that it may trigger specific mechanical sensors that activate the AT1 receptor from inside the cell.
Brenda Riley, Assistant Editor Signaling Gateway
article
Yunzeng Zou, Hiroshi Akazawa, Yingjie Qin, Masanori Sano, Hiroyuki Takano, Tohru Minamino, Noriko Makita, Koji Iwanaga, Weidong Zhu, Sumiyo Kudoh, Haruhiro Toko, Koichi Tamura, Minoru Kihara, Toshio Nagai, Akiyoshi Fukamizu, Satoshi Umemura, Taroh Iiri, Toshiro Fujita & Issei Komuro Mechanical stress activates angiotensin II type 1 receptor without the involvement of angiotensin II Nature Cell Biology, 6, 499 - 506, (June 2004); doi:10.1038/ncb1137
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