scholarly journals Phosphorylation determines the binding of microtubule-associated protein 2 (MAP2) to microtubules in living cells.

1991 ◽  
Vol 114 (4) ◽  
pp. 735-743 ◽  
Author(s):  
B Brugg ◽  
A Matus
Keyword(s):  
Kinase Activity ◽  
Critical Factor ◽  
Living Cells ◽  
Binding Activity ◽  
Protein Kinase Activity ◽  
Phosphatase Inhibitors ◽  
In Vitro Binding ◽  
Vitro Binding ◽  
Phosphate Incorporation

The influence of phosphorylation on the binding of microtubule-associated protein 2 (MAP2) to cellular microtubules was studied by microinjecting MAP2 in various phosphorylation states into rat-1 fibroblasts, which lack endogenous MAP2. Conventionally prepared brain MAP2, containing 10 mol of endogenous phosphate per mol (MAP2-P10), was completely bound to cellular microtubules within 2-3 min after injection. MAP2 prepared in the presence of phosphatase inhibitors, containing 25 mol/mol of phosphate (MAP2-P25), also bound completely. However, MAP2 whose phosphate content had been reduced to 2 mol phosphate per mol by treatment with alkaline phosphatase in vitro (MAP2-P2) did not initially bind to microtubules, suggesting that phosphorylation of certain sites in MAP2 is essential for binding to microtubules. MAP2-P10 was further phosphorylated in vitro via an endogenously bound protein kinase activity, adding 12 more phosphates, giving a total of 22 mol/mol. This preparation (MAP2-P10+12) also did not bind to microtubules. Assay of the binding of these preparations to taxol-stabilized tubulin polymers in vitro confirmed that their binding to tubulin depended on the state of phosphorylation, but the results obtained in microinjection experiments differed in some cases from in vitro binding. The results suggest that the site of phosphate incorporation rather than the amount is the critical factor in determining microtubule binding activity of MAP2. Furthermore, the interaction of MAP2 with cellular microtubules may be influenced by additional factors that are not evident in vitro.

scholarly journals Dynamics of Ankyrin-containing Complexes in Chicken Embryonic Erythroid Cells: Role of Phosphorylation

2001 ◽  
Vol 12 (12) ◽  
pp. 3864-3874 ◽  
Author(s):  
Sourav Ghosh ◽  
John V. Cox
Keyword(s):  
Complex Formation ◽  
Anion Exchanger ◽  
Erythroid Cells ◽  
Binding Studies ◽  
Phosphatase Inhibitors ◽  
In Vitro Binding ◽  
Vitro Binding

Chicken erythroid ankyrin undergoes a fairly rapid cycle of cytoskeletal association, dissociation, and turnover. In addition, the cytoskeletal association of ankyrin is regulated by phosphorylation. Treatment of erythroid cells with serine and threonine phosphatase inhibitors stimulated the hyperphosphorylation of the 225- and 205-kDa ankyrin isoforms, and dissociated the bulk of these isoforms from cytoskeletal spectrin. In vitro binding studies have shown that this dissociation of ankyrin from spectrin in vivo can be attributed to a reduced ability of hyperphosphorylated ankyrin to bind spectrin. Interestingly, a significant fraction of detergent insoluble ankyrin accumulates in a spectrin-independent pool. At least some of this spectrin-independent pool of ankyrin is complexed with the AE1 anion exchanger, and the solubility properties of this pool are also regulated by phosphorylation. Treatment of cells with serine and threonine phosphatase inhibitors had no effect on ankyrin/AE1 complex formation. However, these inhibitors were sufficient to shift ankyrin/AE1 complexes from the detergent insoluble to the soluble pool. These analyses, which are the first to document the in vivo consequences of ankyrin phosphorylation, indicate that erythroid ankyrin-containing complexes can undergo dynamic rearrangements in response to changes in phosphorylation.

Developmental and spatial regulation of a Dictyostelium prespore gene: cis-acting elements and a cAMP-induced, developmentally regulated DNA binding activity

Development ◽  
1991 ◽  
Vol 113 (3) ◽  
pp. 947-958 ◽  
Author(s):  
L. Haberstroh ◽  
J. Galindo ◽  
R.A. Firtel
Keyword(s):  
Point Mutations ◽  
Binding Activity ◽  
Spatial Patterning ◽  
Wild Type ◽  
Developmentally Regulated ◽  
Sequence Element ◽  
In Vitro Binding ◽  
Vitro Binding

Previously, 5′ deletion analysis revealed three important upstream regions within the regulatory region of the cAMP-induced, prespore gene SP60 of D. discoidium, each of which contains a CA-rich sequence element (CAE: consensus CACACAYYYCACACAAA/T). In this study, we have made site-directed mutations within these CAEs and examined their effect on reporter gene activity (luciferase or lacZ). Point mutations within or deletion of the distal CAE (CAE-1), middle CAE (CAE-2) or proximal CAE (CAE-3) result in substantial decreases in promoter activity at 18 h of development or in response to cAMP. lacZ fusions made with the CAE mutant promoters produced novel beta-gal staining patterns that suggest the presence of one or more morphogen gradients within the prespore zone of the slug and indicate that the CAEs are also important in regulating the spatial patterning of SP60 expression in the multicellular aggregate. Gel mobility shift assays were used to identify activities from crude nuclear extracts that bind oligonucleotides containing the CAEs. One of the binding activities is not observed in extracts from vegetative cells or cells in early development and is induced during multicellular development with kinetics similar to those of SP60 gene expression. This activity is also induced in response to cAMP and specifically binds the wild-type CAE-1- and CAE-2-containing oligonucleotides. CAE-1 and CAE-2 oligonucleotides containing point mutations within the CAE core sequence neither bind to nor compete for the cAMP-induced, developmentally regulated factor(s) and result in substantial reductions in expression levels when substituted for the wild-type CAEs in vivo. The correlation between in vitro binding and in vivo function suggests that the CAE-1/CAE-2 binding activity may be involved in regulating cAMP and developmentally induced expression of SP60. A second, constitutive in vitro binding activity with high affinity to CAE-3 is also described. Models are proposed to relate the binding activities with the effects of the mutations on the spatial patterning of SP60-lacZ expression.

Discovery of New AKT1 Inhibitors by Combination of In silico Structure Based Virtual Screening Approaches and Biological Evaluations

2019 ◽  
Author(s):  
Filip Fratev ◽  
Denisse A. Gutierrez ◽  
Renato J. Aguilera ◽  
suman sirimulla
Keyword(s):  
Virtual Screening ◽  
Success Rate ◽  
Protein Interactions ◽  
In Silico ◽  
Biological Data ◽  
In Vitro Binding ◽  
Vitro Binding ◽  
Screening Protocol ◽  
Screening Approaches

AKT1 is emerging as a useful target for treating cancer. Herein, we discovered a new set of ligands that inhibit the AKT1, as shown by in vitro binding and cell line studies, using a newly designed virtual screening protocol that combines structure-based pharmacophore and docking screens. Taking together with the biological data, the combination of structure based pharamcophore and docking methods demonstrated reasonable success rate in identifying new inhibitors (60-70%) proving the success of aforementioned approach. A detail analysis of the ligand-protein interactions was performed explaining observed activities.<br>

scholarly journals ADP-ribosylation of integrin α7 modulates the binding of integrin α7β1 to laminin

2004 ◽  
Vol 385 (1) ◽  
pp. 309-317 ◽  
Author(s):  
Zhefeng ZHAO ◽  
Joanna GRUSZCZYNSKA-BIEGALA ◽  
Anna ZOLKIEWSKA
Keyword(s):  
C2c12 Myotubes ◽  
Post Translational Modification ◽  
Integrin Β1 ◽  
Adp Ribosylation ◽  
In Vitro Binding ◽  
Vitro Binding ◽  
C2c12 Skeletal Muscle Cells ◽  
Adp Ribosyltransferase

The extracellular domain of integrin α7 is ADP-ribosylated by an arginine-specific ecto-ADP-ribosyltransferase after adding exogenous NAD+ to intact C2C12 skeletal muscle cells. The effect of ADP-ribosylation on the structure or function of integrin α7β1 has not been explored. In the present study, we show that ADP-ribosylation of integrin α7 takes place exclusively in differentiated myotubes and that this post-translational modification modulates the affinity of α7β1 dimer for its ligand, laminin. ADP-ribosylation in the 37-kDa ‘stalk’ region of α7 that takes place at micromolar NAD+ concentrations increases the binding of the α7β1 dimer to laminin. Increased in vitro binding of integrin α7β1 to laminin after ADP-ribosylation of the 37-kDa fragment of α7 requires the presence of Mn2+ and it is not observed in the presence of Mg2+. In contrast, ADP-ribosylation of the 63-kDa N-terminal region comprising the ligand-binding site of α7 that occurs at approx. 100 μM NAD+ inhibits the binding of integrin α7β1 to laminin. Furthermore, incubation of C2C12 myotubes with NAD+ increases the expression of an epitope on integrin β1 subunit recognized by monoclonal antibody 9EG7. We discuss our results based on the current models of integrin activation. We also hypothesize that ADP-ribosylation may represent a mechanism of regulation of integrin α7β1 function in myofibres in vivo when the continuity of the membrane is compromised and NAD+ is available as a substrate for ecto-ADP-ribosylation.

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