scholarly journals Differences in the dynamics of the tandem-SH2 modules of the Syk and ZAP-70 tyrosine kinases

2021 ◽  
Author(s):  
Helen T. Hobbs ◽  
Neel H. Shah ◽  
Jean M. Badroos ◽  
Christine L. Gee ◽  
Susan Marqusee ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Tyrosine Kinases ◽  
Bound State ◽  
Kinase Domain ◽  
Sh2 Domain ◽  
Hydrogen Deuterium Exchange ◽  
Inactive Conformation ◽  
Downstream Signaling ◽  
Hydrogen Deuterium ◽  
Dynamics Simulations

The catalytic activity of Syk-family tyrosine kinases is regulated by a tandem-SH2 module (tSH2 module). In the autoinhibited state, this module adopts a conformation which stabilizes an inactive conformation of the kinase domain. The binding of the tSH2 module to doubly-phosphorylated tyrosine-containing motifs necessitates a conformational change, thereby relieving kinase inhibition and promoting activation. We determined the crystal structure of the isolated tSH2 module of Syk and find, in contrast to ZAP-70, that its conformation more closely resembles that of the peptide-bound state, rather than the autoinhibited state. Hydrogen-deuterium exchange by mass spectrometry, as well as molecular dynamics simulations, reveal that the dynamics of the tSH2 modules of Syk and ZAP-70 differ, with most of these differences occurring in the C-terminal SH2 domain. Our data suggest that the conformational landscapes of the tSH2 modules in Syk and ZAP-70 have been tuned differently, such that the auto-inhibited conformation of the Syk tSH2 module is less stable. This feature of Syk likely contributes to its ability to more readily escape autoinhibition when compared to ZAP-70, consistent with tighter control of downstream signaling pathways in T cells.

SHCA PHOSPHOTYROSINE DERIVED SIGNALING IS REQUIRED FOR THE MAINTENANCE OF CARDIAC FUNCTION

2008 ◽  
Vol 31 (4) ◽  
pp. 23
Author(s):  
Rachel Vanderlaan ◽  
Rod Hardy ◽  
Golam Kabir ◽  
Peter Back ◽  
A J Pawson
Keyword(s):  
Extracellular Matrix ◽  
Cardiac Function ◽  
Tyrosine Kinases ◽  
Sh2 Domain ◽  
Scaffolding Protein ◽  
Restricted Domain ◽  
Downstream Signaling ◽  
Matrix Interactions ◽  
Extracellular Matrix Interactions ◽  
Ptb Domain

Background: ShcA, a scaffolding protein, generates signalspecificity by docking to activated tyrosine kinases through distinct phosphotyrosine recognition motifs, while mediating signal complexity through formation of diverse downstream phosphotyrosine complexes. Mammalian ShcA encodes 3 isoforms having a modular architecture of a PTB domain and SH2 domain, separated by a CH1 region containing tyrosine phosphorylation sites important in Ras-MAPK activation. Objective and Methods: ShcA has a necessary role in cardiovascular development^1,2. However, the role of ShcA in the adult myocardium is largely unknown, also unclear, is how ShcA uses its signaling modules to mediate downstream signaling. To this end, cre/loxP technology was employed to generate a conditional ShcA allele series. The myocardial specific ShcA KO (ShcA CKO) and myocardial restricted domain mutant KI mice were generated using cre expressed from the mlc2v locus^3 coupled with the ShcA floxed allele and in combination with the individual ShcA domain mutant KI alleles^2. Results: ShcACKO mice develop a dilated cardiomyopathy phenotype by 3 months of life, typified by depressed cardiac function and enlarged chamber dimensions. Isolated cardiomyocytes from ShcA CKO mice have preserved contractility indicating an uncoupling between global heart function and single myocyte contractile mechanics. Force-length experiments suggest that the loss of shcAmediates the uncoupling through deregulation of extracellular matrix interactions. Subsequent, analysis of the ShcA myocardial restricted domain mutant KImice suggests that ShcA requires PTB domain docking to upstream tyrosine kinases and subsequent phosphorylation of the CH1 tyrosines important for downstream signaling. Conclusion: ShcA is required for proper maintenance of cardiac function, possibly regulation of extracellular matrix interactions. References: 1. Lai KV, Pawson AJ. The ShcA phosphotyrosine docking protein sensitizescardiovascular signaling in the mouse embryo. Genes and Dev 2000;14:1132-45. 2. Hardy WR. et al. Combinatorial ShcA docking interactions supportdiversity in tissue morphogenesis. Science2007;317:251-6. 3.Minamisawa, s. et al. A post-transcriptional compensatory pathway inheterozygous ventricular myosin light chain 2-deficient mice results in lack ofgene dosage effect during normal cardiac growth or hypertrophy. J Biol Chem 1999;274:10066-70.

The Src family kinase Fgr is a transforming oncoprotein that functions independently of SH3-SH2 domain regulation

2018 ◽  
Vol 11 (553) ◽  
pp. eaat5916 ◽  
Author(s):  
Kexin Shen ◽  
Jamie A. Moroco ◽  
Ravi K. Patel ◽  
Haibin Shi ◽  
John R. Engen ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Colony Formation ◽  
Family Members ◽  
Cellular Transformation ◽  
Soft Agar ◽  
Kinase Domain ◽  
Sh2 Domain ◽  
Mass Spectrometry Data ◽  
Activation Loop ◽  
Exchange Mass Spectrometry

Fgr is a member of the Src family of nonreceptor tyrosine kinases, which are overexpressed and constitutively active in many human cancers. Fgr expression is restricted to myeloid hematopoietic cells and is markedly increased in a subset of bone marrow samples from patients with acute myeloid leukemia (AML). Here, we investigated the oncogenic potential of Fgr using Rat-2 fibroblasts that do not express the kinase. Expression of either wild-type or regulatory tail-mutant constructs of Fgr promoted cellular transformation (inferred from colony formation in soft agar), which was accompanied by phosphorylation of the Fgr activation loop, suggesting that the kinase domain of Fgr functions independently of regulation by its noncatalytic SH3-SH2 region. Unlike other family members, recombinant Fgr was not activated by SH3-SH2 domain ligands. However, hydrogen-deuterium exchange mass spectrometry data suggested that the regulatory SH3 and SH2 domains packed against the back of the kinase domain in a Src-like manner. Sequence alignment showed that the activation loop of Fgr was distinct from that of all other Src family members, with proline rather than alanine at the +2 position relative to the activation loop tyrosine. Substitution of the activation loop of Fgr with the sequence from Src partially inhibited kinase activity and suppressed colony formation. Last, Fgr expression enhanced the sensitivity of human myeloid progenitor cells to the cytokine GM-CSF. Because its kinase domain is not sensitive to SH3-SH2–mediated control, simple overexpression of Fgr without mutation may contribute to oncogenic transformation in AML and other blood cancers.

scholarly journals Constitutive signal bias mediated by the human GHRHR splice variant 1

2021 ◽  
Vol 118 (40) ◽  
pp. e2106606118
Author(s):  
Zhaotong Cong ◽  
Fulai Zhou ◽  
Chao Zhang ◽  
Xinyu Zou ◽  
Huibing Zhang ◽  
...  
Keyword(s):  
Splice Variant ◽  
Human Growth ◽  
Bound State ◽  
G Protein Coupled Receptors ◽  
Cancer Cell Proliferation ◽  
Downstream Signaling ◽  
Functional Studies ◽  
Dynamics Simulations ◽  
Protein Molecular Dynamics ◽  
G Protein Coupled

Alternative splicing of G protein–coupled receptors has been observed, but their functions are largely unknown. Here, we report that a splice variant (SV1) of the human growth hormone–releasing hormone receptor (GHRHR) is capable of transducing biased signal. Differing only at the receptor N terminus, GHRHR predominantly activates Gs while SV1 selectively couples to β-arrestins. Based on the cryogenic electron microscopy structures of SV1 in the apo state or GHRH-bound state in complex with the Gs protein, molecular dynamics simulations reveal that the N termini of GHRHR and SV1 differentiate the downstream signaling pathways, Gs versus β-arrestins. As suggested by mutagenesis and functional studies, it appears that GHRH-elicited signal bias toward β-arrestin recruitment is constitutively mediated by SV1. The level of SV1 expression in prostate cancer cells is also positively correlated with ERK1/2 phosphorylation but negatively correlated with cAMP response. Our findings imply that constitutive signal bias may be a mechanism that ensures cancer cell proliferation.

scholarly journals Load-dependent destabilization of the γ-rotor shaft in FOF1 ATP synthase revealed by hydrogen/deuterium-exchange mass spectrometry

2016 ◽  
Vol 113 (9) ◽  
pp. 2412-2417 ◽  
Author(s):  
Siavash Vahidi ◽  
Yumin Bi ◽  
Stanley D. Dunn ◽  
Lars Konermann
Keyword(s):  
Mass Spectrometry ◽  
Molecular Motor ◽  
Conformational Dynamics ◽  
Bound State ◽  
Deuterium Exchange ◽  
Hydrogen Deuterium Exchange ◽  
Exchange Mass Spectrometry ◽  
Hydrogen Deuterium ◽  
Membrane Bound ◽  
Deuterium Exchange Mass Spectrometry

FoF1 is a membrane-bound molecular motor that uses proton-motive force (PMF) to drive the synthesis of ATP from ADP and Pi. Reverse operation generates PMF via ATP hydrolysis. Catalysis in either direction involves rotation of the γε shaft that connects the α3β3 head and the membrane-anchored cn ring. X-ray crystallography and other techniques have provided insights into the structure and function of FoF1 subcomplexes. However, interrogating the conformational dynamics of intact membrane-bound FoF1 during rotational catalysis has proven to be difficult. Here, we use hydrogen/deuterium exchange mass spectrometry to probe the inner workings of FoF1 in its natural membrane-bound state. A pronounced destabilization of the γ C-terminal helix during hydrolysis-driven rotation was observed. This behavior is attributed to torsional stress in γ, arising from γ⋅⋅⋅α3β3 interactions that cause resistance during γ rotation within the apical bearing. Intriguingly, we find that destabilization of γ occurs only when FoF1 operates against a PMF-induced torque; the effect disappears when PMF is eliminated by an uncoupler. This behavior resembles the properties of automotive engines, where bearings inflict greater forces on the crankshaft when operated under load than during idling.

scholarly journals The common src homology region 2 domain of cytoplasmic signaling proteins is a positive effector of v-fps tyrosine kinase function.

1989 ◽  
Vol 9 (10) ◽  
pp. 4131-4140 ◽  
Author(s):  
C A Koch ◽  
M Moran ◽  
I Sadowski ◽  
T Pawson
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Kinase Activity ◽  
Specific Activity ◽  
Intramolecular Interaction ◽  
Kinase Domain ◽  
Sh2 Domain ◽  
Homology Region ◽  
Src Homology ◽  
Type V

A conserved noncatalytic domain SH2 (for src homology region 2) is located immediately N terminal to the kinase domains of all cytoplasmic protein-tyrosine kinases. We found that the wild-type v-fps SH2 domain stimulated the enzymatic activity of the adjacent kinase domain 10-fold and functioned as a powerful positive effector of catalytic and transforming activities within the v-fps oncoprotein (P130gag-fps). Partial proteolysis of P130gag-fps and supporting genetic data indicated that the v-fps SH2 domain exerts its effect on catalytic activity through an intramolecular interaction with the kinase domain. Amino acid alterations in the SH2 domain that impaired kinase function interfered with association of the SH2 domain with the kinase domain. Deletion of a conserved octapeptide motif converted the v-fps SH2 domain from an activator to an inhibitor of tyrosine kinase activity. This latent inhibitory activity of v-fps SH2 has functional implications for phospholipase C-gamma and p21ras GTPase-activating protein, both of which have two distinct SH2 domains suggestive of complex regulation. In addition to regulating the specific activity of the kinase domain, the SH2 domain of P130gag-fps was also found to be required for the tyrosine phosphorylation of specific cellular proteins, notably polypeptides of 124 and 62 kilodaltons. The SH2 domain therefore appears to play a dual role in regulation of kinase activity and recognition of cellular substrates.

scholarly journals Fusion surface structure, function, and dynamics of gamete fusogen HAP2

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Juan Feng ◽  
Xianchi Dong ◽  
Jennifer Pinello ◽  
Jun Zhang ◽  
Chafen Lu ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Structure Function ◽  
Surface Structure ◽  
Outer Surface ◽  
Fold Axis ◽  
Hydrogen Deuterium Exchange ◽  
Apical Portion ◽  
Gamete Fusion ◽  
Hydrophobic Residues ◽  
Hydrogen Deuterium

HAP2 is a class II gamete fusogen in many eukaryotic kingdoms. A crystal structure of Chlamydomonas HAP2 shows a trimeric fusion state. Domains D1, D2.1 and D2.2 line the 3-fold axis; D3 and a stem pack against the outer surface. Surprisingly, hydrogen-deuterium exchange shows that surfaces of D1, D2.2 and D3 closest to the 3-fold axis are more dynamic than exposed surfaces. Three fusion helices in the fusion loops of each monomer expose hydrophobic residues at the trimer apex that are splayed from the 3-fold axis, leaving a solvent-filled cavity between the fusion loops in each monomer. At the base of the two fusion loops, Arg185 docks in a carbonyl cage. Comparisons to other structures, dynamics, and the greater effect on Chlamydomonas gamete fusion of mutation of axis-proximal than axis-distal fusion helices suggest that the apical portion of each monomer could tilt toward the 3-fold axis with merger of the fusion helices into a common fusion surface.

The common src homology region 2 domain of cytoplasmic signaling proteins is a positive effector of v-fps tyrosine kinase function

1989 ◽  
Vol 9 (10) ◽  
pp. 4131-4140
Author(s):  
C A Koch ◽  
M Moran ◽  
I Sadowski ◽  
T Pawson
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Kinase Activity ◽  
Specific Activity ◽  
Intramolecular Interaction ◽  
Kinase Domain ◽  
Sh2 Domain ◽  
Homology Region ◽  
Src Homology ◽  
Type V

A conserved noncatalytic domain SH2 (for src homology region 2) is located immediately N terminal to the kinase domains of all cytoplasmic protein-tyrosine kinases. We found that the wild-type v-fps SH2 domain stimulated the enzymatic activity of the adjacent kinase domain 10-fold and functioned as a powerful positive effector of catalytic and transforming activities within the v-fps oncoprotein (P130gag-fps). Partial proteolysis of P130gag-fps and supporting genetic data indicated that the v-fps SH2 domain exerts its effect on catalytic activity through an intramolecular interaction with the kinase domain. Amino acid alterations in the SH2 domain that impaired kinase function interfered with association of the SH2 domain with the kinase domain. Deletion of a conserved octapeptide motif converted the v-fps SH2 domain from an activator to an inhibitor of tyrosine kinase activity. This latent inhibitory activity of v-fps SH2 has functional implications for phospholipase C-gamma and p21ras GTPase-activating protein, both of which have two distinct SH2 domains suggestive of complex regulation. In addition to regulating the specific activity of the kinase domain, the SH2 domain of P130gag-fps was also found to be required for the tyrosine phosphorylation of specific cellular proteins, notably polypeptides of 124 and 62 kilodaltons. The SH2 domain therefore appears to play a dual role in regulation of kinase activity and recognition of cellular substrates.

scholarly journals Functional importance of stripping in NFκB signaling revealed by a stripping-impaired IκBα mutant

2017 ◽  
Vol 114 (8) ◽  
pp. 1916-1921 ◽  
Author(s):  
Holly E. Dembinski ◽  
Kevin Wismer ◽  
Jesse D. Vargas ◽  
Gajendra W. Suryawanshi ◽  
Nadja Kern ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Bound State ◽  
Hydrogen Deuterium Exchange ◽  
Functional Importance ◽  
Exchange Mass Spectrometry ◽  
Turn On ◽  
Hydrogen Deuterium ◽  
Transcription Sites ◽  
Deuterium Exchange Mass Spectrometry

Stress-response transcription factors such as NFκB turn on hundreds of genes and must have a mechanism for rapid cessation of transcriptional activation. We recently showed that the inhibitor of NFκB signaling, IκBα, dramatically accelerates the dissociation of NFκB from transcription sites, a process we have called “stripping.” To test the role of the IκBα C-terminal PEST (rich in proline, glutamic acid, serine, and threonine residues) sequence in NFκB stripping, a mutant IκBα was generated in which five acidic PEST residues were mutated to their neutral analogs. This IκBα(5xPEST) mutant was impaired in stripping NFκB from DNA and formed a more stable intermediate ternary complex than that formed from IκBα(WT) because DNA dissociated more slowly. NMR and amide hydrogen–deuterium exchange mass spectrometry showed that the IκBα(5xPEST) appears to be “caught in the act of stripping” because it is not yet completely in the folded and NFκB-bound state. When the mutant was introduced into cells, the rate of postinduction IκBα-mediated export of NFκB from the nucleus decreased markedly.

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