Solution structure of the C-terminal SH2 domain of the p85α regulatory subunit of phosphoinositide 3-kinase 1 1Edited by P. E. Wright

1998 ◽  
Vol 276 (2) ◽  
pp. 461-478 ◽  
Author(s):  
Gregg Siegal ◽  
Ben Davis ◽  
Søren M. Kristensen ◽  
Andrew Sankar ◽  
Jeffrey Linacre ◽  
...  
Keyword(s):  
Solution Structure ◽  
Sh2 Domain ◽  
Regulatory Subunit ◽  
Phosphoinositide 3 Kinase

scholarly journals v-Crk Activates the Phosphoinositide 3-Kinase/AKT Pathway by Utilizing Focal Adhesion Kinase and H-Ras

2002 ◽  
Vol 22 (20) ◽  
pp. 7015-7023 ◽  
Author(s):  
Tsuyoshi Akagi ◽  
Kazutaka Murata ◽  
Tomoyuki Shishido ◽  
Hidesaburo Hanafusa
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Critical Role ◽  
Sh3 Domain ◽  
Sh2 Domain ◽  
Dominant Negative ◽  
Regulatory Subunit ◽  
Akt Pathway ◽  
Dominant Negative Mutant ◽  
Phosphoinositide 3 Kinase

ABSTRACT v-Crk, an oncogene product of avian sarcoma virus CT10, efficiently transforms chicken embryo fibroblasts (CEF). We have recently reported that constitutive activation of the phosphoinositide 3-kinase (PI3K)/AKT pathway plays a critical role in the v-Crk-induced transformation of CEF. In the present study we investigated the molecular mechanism by which v-Crk activates the PI3K/AKT pathway. First, we found that v-Crk promotes the association of the p85 regulatory subunit of PI3K with focal adhesion kinase (FAK) by inducing the phosphorylation of the Y397 residue in FAK. This FAK phosphorylation needs activation of the Src family tyrosine kinase(s) for which the v-Crk SH2 domain is responsible. v-Crk was unable to activate the PI3K/AKT pathway in FAK-null cells, indicating the functional importance of FAK. In addition, we found that H-Ras is also required for the activation of the PI3K/AKT pathway. The v-Crk-induced activation of AKT was greatly enhanced by the overexpression of H-Ras or its guanine nucleotide exchange factor mSOS, which binds to the v-Crk SH3 domain, whereas a dominant-negative mutant of H-Ras almost completely suppressed this activation. Furthermore, we showed that v-Crk stimulates the interaction of H-Ras with the Ras binding domain in the PI3K p110 catalytic subunit. Our data indicated that the v-Crk-induced activation of PI3K/AKT pathway was cooperatively achieved by two distinct interactions. One is the interaction of p85 with tyrosine-phosphorylated FAK promoted by the v-Crk SH2 domain, and another is the interaction of p110 with H-Ras dictated by the v-Crk SH3 domain.

scholarly journals Cancer-associated mutations in the p85α N-terminal SH2 domain activate a spectrum of receptor tyrosine kinases

2021 ◽  
Vol 118 (37) ◽  
pp. e2101751118
Author(s):  
Xinran Li ◽  
Amy Y. T. Lau ◽  
Angel S. N. Ng ◽  
Abdullah Aldehaiman ◽  
Yuan Zhou ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Sh2 Domain ◽  
Regulatory Subunit ◽  
Driver Mutations ◽  
Akt Pathway ◽  
Independent Manner ◽  
Inhibitory Interaction ◽  
Her Family ◽  
Protein Levels ◽  
Phosphoinositide 3 Kinase

The phosphoinositide 3-kinase regulatory subunit p85α is a key regulator of kinase signaling and is frequently mutated in cancers. In the present study, we showed that in addition to weakening the inhibitory interaction between p85α and p110α, a group of driver mutations in the p85α N-terminal SH2 domain activated EGFR, HER2, HER3, c-Met, and IGF-1R in a p110α-independent manner. Cancer cells expressing these mutations exhibited the activation of p110α and the AKT pathway. Interestingly, the activation of EGFR, HER2, and c-Met was attributed to the ability of driver mutations to inhibit HER3 ubiquitination and degradation. The resulting increase in HER3 protein levels promoted its heterodimerization with EGFR, HER2, and c-Met, as well as the allosteric activation of these dimerized partners; however, HER3 silencing abolished this transactivation. Accordingly, inhibitors of either AKT or the HER family reduced the oncogenicity of driver mutations. The combination of these inhibitors resulted in marked synergy. Taken together, our findings provide mechanistic insights and suggest therapeutic strategies targeting a class of recurrent p85α mutations.

scholarly journals Structural insights into phosphoinositide 3-kinase activation by the influenza A virus NS1 protein

2010 ◽  
Vol 107 (5) ◽  
pp. 1954-1959 ◽  
Author(s):  
Benjamin G. Hale ◽  
Philip S. Kerry ◽  
David Jackson ◽  
Bernard L. Precious ◽  
Alexander Gray ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Coiled Coil ◽  
Sh2 Domain ◽  
Regulatory Subunit ◽  
Ns1 Protein ◽  
Influenza A Viruses ◽  
Phosphoinositide 3 Kinase ◽  
Α Helix

Seasonal epidemics and periodic worldwide pandemics caused by influenza A viruses are of continuous concern. The viral nonstructural (NS1) protein is a multifunctional virulence factor that antagonizes several host innate immune defenses during infection. NS1 also directly stimulates class IA phosphoinositide 3-kinase (PI3K) signaling, an essential cell survival pathway commonly mutated in human cancers. Here, we present a 2.3-Å resolution crystal structure of the NS1 effector domain in complex with the inter-SH2 (coiled-coil) domain of p85β, a regulatory subunit of PI3K. Our data emphasize the remarkable isoform specificity of this interaction, and provide insights into the mechanism by which NS1 activates the PI3K (p85β:p110) holoenzyme. A model of the NS1:PI3K heterotrimeric complex reveals that NS1 uses the coiled-coil as a structural tether to sterically prevent normal inhibitory contacts between the N-terminal SH2 domain of p85β and the p110 catalytic subunit. Furthermore, in this model, NS1 makes extensive contacts with the C2/kinase domains of p110, and a small acidic α-helix of NS1 sits adjacent to the highly basic activation loop of the enzyme. During infection, a recombinant influenza A virus expressing NS1 with charge-disruption mutations in this acidic α-helix is unable to stimulate the production of phosphatidylinositol 3,4,5-trisphosphate or the phosphorylation of Akt. Despite this, the charge-disruption mutations in NS1 do not affect its ability to interact with the p85β inter-SH2 domain in vitro. Overall, these data suggest that both direct binding of NS1 to p85β (resulting in repositioning of the N-terminal SH2 domain) and possible NS1:p110 contacts contribute to PI3K activation.

scholarly journals Binding of Influenza A Virus NS1 Protein to the Inter-SH2 Domain of p85β Suggests a Novel Mechanism for Phosphoinositide 3-Kinase Activation

2007 ◽  
Vol 283 (3) ◽  
pp. 1372-1380 ◽  
Author(s):  
Benjamin G. Hale ◽  
Ian H. Batty ◽  
C. Peter Downes ◽  
Richard E. Randall
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Rna Binding ◽  
Regulatory Element ◽  
Coiled Coil ◽  
Sh2 Domain ◽  
Regulatory Subunit ◽  
Ns1 Protein ◽  
Phosphoinositide 3 Kinase

Influenza A virus NS1 protein stimulates host-cell phosphoinositide 3-kinase (PI3K) signaling by binding to the p85β regulatory subunit of PI3K. Here, in an attempt to establish a mechanism for this activation, we report further on the functional interaction between NS1 and p85β. Complex formation was found to be independent of NS1 RNA binding activity and is mediated by the C-terminal effector domain of NS1. Intriguingly, the primary direct binding site for NS1 on p85β is the inter-SH2 domain, a coiled-coil structure that acts as a scaffold for the p110 catalytic subunit of PI3K. In vitro kinase activity assays, together with protein binding competition studies, reveal that NS1 does not displace p110 from the inter-SH2 domain, and indicate that NS1 can form an active heterotrimeric complex with PI3K. In addition, it was established that residues at the C terminus of the inter-SH2 domain are essential for mediating the interaction between p85β and NS1. Equivalent residues in p85α have previously been implicated in the basal inhibition of p110. However, such p85α residues were unable to substitute for those in p85β with regards NS1 binding. Overall, these data suggest a model by which NS1 activates PI3K catalytic activity by masking a normal regulatory element specific to the p85β inter-SH2 domain.

Crystal structure of the C-terminal SH2 domain of the p85α regulatory subunit of phosphoinositide 3-kinase: an SH2 domain mimicking its own substrate

1999 ◽  
Vol 292 (4) ◽  
pp. 763-770 ◽  
Author(s):  
Flip J Hoedemaeker ◽  
Gregg Siegal ◽  
S.Mark Roe ◽  
Paul C Driscoll ◽  
Jan Pieter Abrahams
Keyword(s):  
Crystal Structure ◽  
Sh2 Domain ◽  
Regulatory Subunit ◽  
Phosphoinositide 3 Kinase

scholarly journals Fission Yeast Rad26 Is a Regulatory Subunit of the Rad3 Checkpoint Kinase

2002 ◽  
Vol 13 (2) ◽  
pp. 480-492 ◽  
Author(s):  
Tom D. Wolkow ◽  
Tamar Enoch
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Fission Yeast ◽  
Complex Analysis ◽  
Kinase Activity ◽  
Regulatory Subunit ◽  
Kinase Activation ◽  
Checkpoint Proteins ◽  
Cycle Arrest ◽  
Phosphoinositide 3 Kinase

Fission yeast Rad3 is a member of a family of phosphoinositide 3-kinase -related kinases required for the maintenance of genomic stability in all eukaryotic cells. In fission yeast, Rad3 regulates the cell cycle arrest and recovery activities associated with the G2/M checkpoint. We have developed an assay that directly measures Rad3 kinase activity in cells expressing physiological levels of the protein. Using the assay, we demonstrate directly that Rad3 kinase activity is stimulated by checkpoint signals. Of the five other G2/M checkpoint proteins (Hus1, Rad1, Rad9, Rad17, and Rad26), only Rad26 was required for Rad3 kinase activity. Because Rad26 has previously been shown to interact constitutively with Rad3, our results demonstrate that Rad26 is a regulatory subunit, and Rad3 is the catalytic subunit, of the Rad3/Rad26 kinase complex. Analysis of Rad26/Rad3 kinase activation in rad26.T12, a mutant that is proficient for cell cycle arrest, but defective in recovery, suggests that these two responses to checkpoint signals require quantitatively different levels of kinase activity from the Rad3/Rad26 complex.

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