Phosphopeptide binding to the N‐terminal SH2 domain of the p85α subunit of PI 3′‐kinase: A heteronuclear NMR study

Hensmann, Meike; Booker, Grant W.; Panayotou, George; Boyd, Jonathan; Linacre, Jeffrey; Waterfield, Mike; Campbell, Iain D.

doi:10.1002/pro.5560030704

Cited by 47 publications

(45 citation statements)

References 32 publications

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“…5, model b). NMR and crystallographic studies on the isolated p85 nSH2 domain show relatively small conformational changes on phosphopeptide binding (13,14,36). However, in the phosphatase SHP-2, phosphopeptide binding to the SH2 domains causes similarly small conformational changes that are nonetheless sufficient to disinhibit catalytic activity (37).…”

Section: Discussionmentioning

confidence: 99%

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The structure of the inter-SH2 domain of class IA phosphoinositide 3-kinase determined by site-directed spin labeling EPR and homology modeling

Aronoff-Spencer

Backer

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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Phosphoinositide (PI) 3-kinases catalyze the phosphorylation of the D3 position of the inositol ring of PI, and its phosphorylated derivatives and play important roles in many intracellular signal transducing pathways. Class IA PI3-kinases contain distinct regulatory (p85) and catalytic (p110) subunits. p110 is stabilized and inhibited by constitutive association with p85, and is disinhibited when the SH2 domains of p85 bind to tyrosyl-phosphorylated proteins. Because the two subunits do not dissociate, disinhibition of p110 presumably occurs by an allosteric mechanism. To explore the means by which p85 regulates the activity of p110, structures of the inter-SH2 domain of p85 were determined with and without phosphopeptide by using a combination of site directed spin labeling EPR and homology modeling and molecular dynamics. The inter-SH2 domain is assigned as a rigid anti-parallel coiled-coil whose primary function is to bind p110, facilitating inhibition of p110 by the N-terminal SH2 domain of p85.nitroxide ͉ distance measurement ͉ SDSL-EPR ͉ p85 ͉ p110 P hosphoinositide (PI) 3-kinases are important regulators of proliferation, apoptosis, motility, and vesicular trafficking (1). Class IA PI3-kinases contain distinct regulatory (p85) and catalytic (p110) subunits. It has been shown previously that p85 plays two roles with regard to regulation of p110: stabilization of p110 against thermal denaturation and degradation, and inhibition of p110 catalytic activity (2). The N-terminal half of p85 contains an SH3 domain and a Rac͞CDC42-binding domain, which is flanked by two proline-rich domains (3, 4). These domains are important for cytoskeletal regulation but not mitogenic signaling (5), and are not present in three of the regulatory subunit variants (p50␣, p55␣, and p55␥). The Cterminal half of p85 contains two src homology 2 (SH2) domains (the N-terminal or nSH2 domain and the C-terminal or cSH2 domain) separated by the inter-SH2 (iSH2) domain, a putative coiled-coil that contains the binding site for the p110 catalytic subunit (6). Although CD studies of the iSH2 domain show it to be highly helical (7), its structure has not been further characterized.The binding of tyrosine-phosphorylated proteins or peptides to the SH2 domains of p85 releases its inhibition of p110, leading to an activation of p85͞p110 heterodimers (8, 9) through unknown conformational changes. The minimal fragment of p85 capable of regulating p110 is the nSH2 domain linked to the iSH2 domain (10) (Fig. 1a). Although the iSH2 domain is sufficient to bind p110 (6,11,12) it has minimal effects on p110 activity (10). In contrast, the nSH2-iSH2 fragment (heretofore referred to as p85ni) inhibits p110, and p85ni͞p110 complexes are activated on tyrosine phosphopeptide binding. Given that the nSH2 domain is the sole phosphopeptide binding domain in p85ni, several explanations for inhibition͞disinhibition regulation are possible. These include phosphopeptide-induced conformational changes in the nSH2 domain, the iSH2 domain, the adjoining segment, or ...

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Section: Discussionmentioning

confidence: 99%

“…The structure of the isolated nSH2 domain has been determined by solution NMR and x-ray crystallography (13,14). The present work therefore focuses on the structure of the iSH2 domain of p85ni, which has not been previously determined.…”

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confidence: 99%

The structure of the inter-SH2 domain of class IA phosphoinositide 3-kinase determined by site-directed spin labeling EPR and homology modeling

Aronoff-Spencer

Backer

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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“…The p85 N-terminal SH2 (NSH2) domain and PLC-␥1 C-terminal SH2 (CSH2) domain were selected to study the importance of the ␤D5 residue for SH2 domain specificity because their three-dimensional structures, with associated high affinity phosphopeptides have been solved (8,13). 2 The wild-type p85 NSH2 domain contains a Ile (Ile-383) residue at the ␤D5 position and preferentially binds the sequence Tyr(P)-Met-X-Met (2) (Fig.…”

Section: Phosphopeptide Selectivity Of Mutant Phosphoinositide 3-kinamentioning

confidence: 99%

A Single Point Mutation Switches the Specificity of Group III Src Homology (SH) 2 Domains to That of Group I SH2 Domains

Songyang

Gish

Mbamalu

et al. 1995

Journal of Biological Chemistry

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Stimulation of cellular responses by growth factors and cytokines is often accomplished by the activation of protein-tyrosine kinases (1, 6). One of the major consequences of tyrosine phosphorylation is to induce a specific set of protein-protein interactions, and thereby initiate a series of intracellular signaling cascades. The SH2 1 domains of cytosolic signaling proteins mediate the assembly of such complexes by binding to phosphotyrosine moieties within specific sequence contexts (1, 6, 7). Therefore, in order to understand signal transduction by protein-tyrosine kinases, it is important to decode the mechanisms by which SH2 domains achieve specificity in their recognition of phosphotyrosine sites.The specificity of SH2 domains was first systematically studied using a degenerate phosphopeptide library (2, 3). Subsequently, crystal structures of SH2 domains complexed with their high affinity ligands were obtained, providing a structural basis for phosphopeptide recognition by SH2 domains (4,5,8,9). From these studies and related experiments analyzing the in vivo binding sites of various SH2 domains, it has become evident that 3-6 residues C-terminal of phosphotyrosine dictate the specificity of SH2 domain binding. Importantly, SH2 domains can be divided into four subgroups on the basis of their specificity and primary sequences (2). For example, Group I SH2 domains (SH2 domains of Src family, Abl, Crk, GRB2, Nck SH2 domains, etc.) select the general motif Tyr(P)-hydrophilichydrophilic-hydrophobic and have an aromatic amino acid at the ␤D5 position. However, Group III SH2 domains (e.g. SH2 domains of phosphoinositide 3-kinase p85, phospholipase C-␥, and Syp/SHPTP2) select the general motif Tyr(P)-hydrophobic-X-hydrophobic and have Ile or Cys at the ␤D5 position (Fig. 1A). In the three-dimensional structures of Src and Lck SH2 domains, the ␤D5 Tyr is at the surface and makes contacts with the side chains of both the pYϩ1 and pYϩ3 residues of the bound phosphopeptide (4, 5). However, in the three-dimensional structures of Syp and PLC-␥ SH2 domains (Group III), the aliphatic residue at the ␤D5 position is buried deeper in the protein, opening a hydrophobic cavity for the pYϩ1 through pYϩ6 residues (8, 9) (Fig. 1B). To address the importance of the ␤D5 position, we have investigated the effect of substituting this residue on the specificity of two Group III SH2 domains. MATERIALS AND METHODSMutant SH2 Domain Construction-The point mutation (I383Y) on phosphoinositide 3-kinase p85 N-terminal SH2 domain was achieved by PCR mutagenesis using human p85 cDNA as template. The following primers were synthesized: 1, 5Ј-GACGGATCCTTACAAAATGCTGAA-3Ј; 2, 5Ј-GTCGAATTCCATCTTCTTTGACAACTTG-3Ј; 3, 5Ј-GGAAAT-AACAAATTATACAAAATATTTCATCGA-3Ј; 4, 5Ј-TCGATGAAATAT-TTTGTATAATTTGTTATTTCC-3Ј. Two PCR fragments were first produced using primer pairs 1 and 4, and 2 and 3. The mutant cDNA was obtained by ligating these two fragments using PCR with primers 1 and 2. The final PCR products were cut with BamHI and EcoRI, and cloned into pGEX2T v...

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“…The HSQC spectra were acquired with 16 scans, 64 increments in t 1 , and sweep widths of 10000 Hz ( 1 H) and 2400 Hz ( 15 N). Threedimensional 15 N-1 H HSQC total correlation spectroscopy and nuclear Overhauser effect spectroscopy experiments were recorded to verify the published resonance assignments for the N-SH2 domain (31,32).…”

Section: Nmr Spectroscopy Experimentsmentioning

confidence: 97%

Structural and Biochemical Evaluation of the Interaction of the Phosphatidylinositol 3-Kinase p85α Src Homology 2 Domains with Phosphoinositides and Inositol Polyphosphates

Surdo¹,

Bottomley²,

Arcaro³

et al. 1999

Journal of Biological Chemistry

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Src homology 2 (SH2) domains exist in many intracellular proteins and have well characterized roles in signal transduction. SH2 domains bind to phosphotyrosine (Tyr(P))-containing proteins. Although tyrosine phosphorylation is essential for protein-SH2 domain interactions, the binding specificity also derives from sequences C-terminal to the Tyr(P) residue. The high affinity and specificity of this interaction is critical for precluding aberrant cross-talk between signaling pathways. The p85␣ subunit of phosphoinositide 3-kinase (PI 3-kinase) contains two SH2 domains, and it has been proposed that in competition with Tyr(P) binding they may also mediate membrane attachment via interactions with phosphoinositide products of PI 3-kinase. We used nuclear magnetic resonance spectroscopy and biosensor experiments to investigate interactions between the p85␣ SH2 domains and phosphoinositides or inositol polyphosphates. We reported previously a similar approach when demonstrating that some pleckstrin homology domains show binding specificity for distinct phosphoinositides (Salim, K., Bottomley, M. J., Querfurth, E., Zvelebil, M. J., Gout, I., Scaife, R., Margolis, R. L., Gigg, R., Smith, C. I., Driscoll, P. C., Waterfield, M. D., and Panayotou, G. (1996) EMBO J. 15, 6241-6250). However, neither SH2 domain exhibited binding specificity for phosphoinositides in phospholipid bilayers. We show that the p85␣ SH2 domain Tyr(P) binding pockets indiscriminately accommodate phosphoinositides and inositol polyphosphates. Binding of the SH2 domains to Tyr(P) peptides was only poorly competed for by phosphoinositides or inositol polyphosphates. We conclude that these ligands do not bind p85␣ SH2 domains with high affinity or specificity. Moreover, we observed that although wortmannin blocks PI 3-kinase activity in vivo, it does not affect the ability of tyrosinephosphorylated proteins to bind to p85␣. Consequently phosphoinositide products of PI 3-kinase are unlikely to regulate signaling through p85␣ SH2 domains.

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Phosphopeptide binding to the N‐terminal SH2 domain of the p85α subunit of PI 3′‐kinase: A heteronuclear NMR study

Cited by 47 publications

References 32 publications

The structure of the inter-SH2 domain of class IA phosphoinositide 3-kinase determined by site-directed spin labeling EPR and homology modeling

The structure of the inter-SH2 domain of class IA phosphoinositide 3-kinase determined by site-directed spin labeling EPR and homology modeling

A Single Point Mutation Switches the Specificity of Group III Src Homology (SH) 2 Domains to That of Group I SH2 Domains

Structural and Biochemical Evaluation of the Interaction of the Phosphatidylinositol 3-Kinase p85α Src Homology 2 Domains with Phosphoinositides and Inositol Polyphosphates

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