The Solution Structure and Dynamics of the Pleckstrin Homology Domain of G Protein-coupled Receptor Kinase 2 (β-Adrenergic Receptor Kinase 1)

Fushman, David; Najmabadi-Haske, Taraneh; Cahill, Sean M.; Zheng, Jie; LeVine, Harry; Cowburn, David

doi:10.1074/jbc.273.5.2835

Cited by 76 publications

(69 citation statements)

References 56 publications

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“…This region exhibits similarity to the basic residues in the extended PH domain of GRK2, i.e. the molten helix, involved in interaction with G␤␥ (28,29). Other studies have illustrated the importance of sequence in the Y-domain of PLC-␤2 in G␤␥-promoted activation (39), and therefore the precise structural determinants for activation of PLC-␤ isozymes by G␤␥ remain to be resolved.…”

Section: Discussionmentioning

confidence: 99%

“…This ␣-helical extension is rich in arginine and lysine residues and thus reminiscent of the highly mobile, positively charged "molten helix" observed in the NMR structure of the human GRK2 PH domain (PDB code 1bak; Fig. 1B) and critical for GRK2 binding to G-protein ␤␥ dimers (28,29).…”

Section: Fig 1 Domain Organization Of Plc Isozymes and Secondary Stmentioning

confidence: 99%

“…B, multiple sequence alignment of the PH domains of rat PLC-␦1 (SwissProt PID1 Rat), rat and human PLC-⑀ (GenBank TM accession numbers AF323615 and AF190642), C. elegans PLC210 (GenBank TM AF044576), and human GRK2 (GenBank TM U08438). Known secondary structures within the PH domains of rat PLC-␦1 (26) and GRK2 (28) are overlined and underlined, respectively. The term molten-helix refers to a highly mobile extension of the PH domain COOH-terminal ␣-helix (28) required for GRK2 binding to G␤␥ (29).…”

Section: Fig 1 Domain Organization Of Plc Isozymes and Secondary Stmentioning

confidence: 99%

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Activation of Phospholipase C-ε by Heterotrimeric G Protein βγ-Subunits

Wing

Houston

Kelley

et al. 2001

Journal of Biological Chemistry

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PLC-⑀ was identified recently as a phosphoinositidehydrolyzing phospholipase C (PLC) containing catalytic domains (X, Y, and C2) common to all PLC isozymes as well as unique CDC25-and Ras-associating domains. Novel regulation of this PLC isozyme by the Ras oncoprotein and ␣-subunits (G␣ 12 ) of heterotrimeric G proteins was illustrated. Sequence analyses of PLC-⑀ revealed previously unrecognized PH and EF-hand domains in the amino terminus. The known interaction of G␤␥ subunits with the PH domains of other proteins led us to examine the capacity of G␤␥ to activate PLC-⑀.

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Section: Discussionmentioning

confidence: 99%

Section: Fig 1 Domain Organization Of Plc Isozymes and Secondary Stmentioning

confidence: 99%

Section: Fig 1 Domain Organization Of Plc Isozymes and Secondary Stmentioning

confidence: 99%

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Activation of Phospholipase C-ε by Heterotrimeric G Protein βγ-Subunits

Wing

Houston

Kelley

et al. 2001

Journal of Biological Chemistry

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“…These experiments were performed at five magnetic fields of 9.4, 11.7, 14.1, 16.4, and 18.8 Tesla, and used standard pulse sequences (e.g. 33). The NOEs were determined using a flip-back measurement scheme 34 for water suppression, the recycling delay was 4-5 s (see Supporting Table 1 for complete list of relaxation delays).…”

Section: Sample Preparation and Nmr Measurementsmentioning

confidence: 99%

Variability of the ¹⁵N Chemical Shielding Tensors in the B3 Domain of Protein G from ¹⁵N Relaxation Measurements at Several Fields. Implications for Backbone Order Parameters

2006

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We applied a combination of 15 N relaxation and CSA/dipolar cross-correlation measurements at five magnetic fields (9.4, 11.7, 14.1, 16.4, and 18.8 Tesla) to determine the 15 N chemical shielding tensors for backbone amides in protein G in solution. The data were analyzed using various modelindependent approaches and those based on Lipari-Szabo approximation, all of them yielding similar results. The results indicate a range of site-specific values of the anisotropy (CSA) and orientation of the 15 N chemical shielding tensor, similar to those in ubiquitin. Assuming a Gaussian distribution of the 15 N CSA values, the mean anisotropy is -173.9 to -177.2 ppm (for 1.02-Å NH-bond length) and the site-so-site CSA variability is ±17.6 to ±21.4 ppm, depending on the method used. This CSA variability is significantly larger than derived previously for ribonuclease H or recently, using "metaanalysis" for ubiquitin. Standard interpretation of 15 N relaxation studies of backbone dynamics in proteins involves an a priori assumption of a uniform 15 N CSA. We show that this assumption leads to a significant discrepancy between the order parameters obtained at different fields. Using the sitespecific CSAs obtained from our study removes this discrepancy and allows simultaneous fit of relaxation data at all five fields to Lipari-Szabo spectral densities. These findings emphasize the necessity of taking into account the variability of 15 N CSA for accurate analysis of protein dynamics from 15 N relaxation measurements.

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“…For these reasons, we initially analyzed a relatively large construct of the mDvl1 DEP domain, mDvl1(390-498), to ensure that all of the residues of the domain were present in a single construct. After completing the backbone assignments of this construct, heteronuclear 15 N-1 H nuclear Overhauser effects (NOEs) were measured using standard methods 30 . The results indicated that residues 402-495 are well folded and that the Nterminal 10 amino acids is unstructured.…”

Section: Boundaries Of the Dep Domain Of Mdvl1mentioning

confidence: 99%

Untitled

et al. 2000

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The DEP domain of Dishevelled (Dvl) proteins transduces signals to effector proteins downstream of Dvl in the Wnt pathway. Here we report that DEP-containing mutants inhibit Wnt-induced, but not Dvl-induced, activation of the transcription factor Lef-1. This inhibitory effect is weakened by a K434M mutation. Nuclear magnetic resonance spectroscopy revealed that the DEP domain of mouse Dvl1 comprises a three-helix bundle, a β-hairpin 'arm' and two short β-strands at the Cterminal region. Lys 434 is located at the tip of the β-hairpin 'arm'. Based on our findings, we conclude that DEP interacts with regulators upstream of Dvl via a strong electric dipole on the molecule's surface created by Lys 434, Asp 445 and Asp 448; the electric dipole and the putative membrane binding site are at two different locations.The Wnt signaling pathway is a key regulatory pathway for cellular development and growth. Wnt signaling has been extensively studied in Drosophila, Caenorhabditis elegans, Xenopus, and mammalian systems [1][2][3][4][5] . Through genetic studies, a working model of the Wnt signaling pathway has been established. Secreted Wnt proteins bind to Frizzled transmembrane receptors. The receptor-ligand complex activates the Dishevelled (Dsh and Dvl) proteins, which suppress the activity of glycogen synthase kinase-3β (GSK-3β). If its activity is not suppressed, GSK-3β binds to adenomatous polyposis coli (APC) protein and axin and then phosphorylates specific Ser and Thr residues at the N-terminus of β-catenin. In turn, the ubiquitin-proteasome pathway rapidly degrades hyperphosphorylated β-catenin. Wnt activation prevents this process by promoting the cytosolic accumulation and subsequent translocation of β-catenin into the nucleus. Once in the nucleus, β-catenin binds to the transcription factors T-cell factor (Tcf) or lymphoid enhancer factor (Lef) and acts as a transcriptional coactivator 5 . The result is increased expression of Tcf or Lef regulated target genes, such as Myc 6 and the gene encoding cyclin D 7 .© 2000 Nature America Inc. Correspondence should be addressed to: J.Z. Jie.Zheng@stjude.org. HHS Public Access DEP domain interacts with upstream regulators of DvlWe and others have demonstrated that Wnt-1 or Dvl1, when expressed in NIH3T3 cells, can activate the transcription regulator 16,28,29). This activation depends on β-catenin and is suppressed by GSK-3β and axin. To characterize the role of the mDvl1 DEP domain in Wnt signaling, we examined the effect of the Dvl1 N-terminal truncation mutant, DvlC1, on Wnt-induced activation of the transcription factor Lef-1. In Lef dependent reporter gene assays, DvlC1 inhibited Wnt-1 induced Lef-1 activation but not Dvl1 induced Lef-1 activation (Fig. 2). To further characterize the specific sequences that inhibit the Wnt-induced activation, we generated two additional mutants of mDvl1: DvlDEP, which contains only the DEP domain; and DvlC2, which encompasses the sequence downstream of the DEP domain. When expressed in NIH3T3 cells, DvlDEP, like DvlC1, bloc...

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The Solution Structure and Dynamics of the Pleckstrin Homology Domain of G Protein-coupled Receptor Kinase 2 (β-Adrenergic Receptor Kinase 1)

Cited by 76 publications

References 56 publications

Activation of Phospholipase C-ε by Heterotrimeric G Protein βγ-Subunits

Activation of Phospholipase C-ε by Heterotrimeric G Protein βγ-Subunits

Variability of the ¹⁵N Chemical Shielding Tensors in the B3 Domain of Protein G from ¹⁵N Relaxation Measurements at Several Fields. Implications for Backbone Order Parameters

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The Solution Structure and Dynamics of the Pleckstrin Homology Domain of G Protein-coupled Receptor Kinase 2 (β-Adrenergic Receptor Kinase 1)

Cited by 76 publications

References 56 publications

Activation of Phospholipase C-ε by Heterotrimeric G Protein βγ-Subunits

Activation of Phospholipase C-ε by Heterotrimeric G Protein βγ-Subunits

Variability of the 15N Chemical Shielding Tensors in the B3 Domain of Protein G from 15N Relaxation Measurements at Several Fields. Implications for Backbone Order Parameters

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Variability of the ¹⁵N Chemical Shielding Tensors in the B3 Domain of Protein G from ¹⁵N Relaxation Measurements at Several Fields. Implications for Backbone Order Parameters