Guanine Nucleotide Binding Properties of Rac2 Mutant Proteins and Analysis of the Responsiveness to Guanine Nucleotide Dissociation Stimulator†,⊥

Biochemistry ◽  
1997 ◽  
Vol 36 (3) ◽  
pp. 626-632 ◽  
Author(s):  
Xuemin Xu ◽  
Yan Wang ◽  
David C. Barry ◽  
Stephen J. Chanock ◽  
Gary M. Bokoch
Keyword(s):  
Nucleotide Binding ◽  
Guanine Nucleotide ◽  
Binding Properties ◽  
Mutant Proteins ◽  
Guanine Nucleotide Binding

scholarly journals Guanine nucleotide binding properties of rap1 purified from human neutrophils

1990 ◽  
Vol 267 (2) ◽  
pp. 407-411 ◽  
Author(s):  
G M Bokoch ◽  
L A Quilliam
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Nucleotide Binding ◽  
Human Neutrophils ◽  
Guanine Nucleotide ◽  
Binding Properties ◽  
Bivalent Cation ◽  
Guanine Nucleotide Binding ◽  
Kinase A

The guanine nucleotide binding properties of rap1 protein purified from human neutrophils were examined using both the protein kinase A-phosphorylated and the non-phosphorylated forms of the protein. Binding of GTP[S] (guanosine 5′-[gamma-thio]triphosphate) or GDP was found to be slow in the presence of free Mg2+, but very rapid in the absence of Mg2+. The binding of guanine nucleotides was found to correlate with the loss of endogenous nucleotide from the rap1 protein, which was rapid in the absence of Mg2+. The relative affinities of GTP and GDP for the binding site on rap1 were modulated by the presence of Mg2+, with a preferential affinity (approx. 15-fold) for GTP observed only in the absence of this bivalent cation. The dissociation of GDP from rap1 was not affected by the G-protein beta/gamma-subunit complex. Phosphorylation of rap1 in vitro by protein kinase A did not modify any of the observed nucleotide-binding parameters. Furthermore, the ability of a cytosolic rap1 GTPase-activating protein to stimulate neutrophil rap1 GTP hydrolysis was not modified by phosphorylation. These data suggest that the activation of rap in vivo may be regulated by the release of endogenous GDP, but that phosphorylation by protein kinase A does not affect guanine nucleotide binding or hydrolysis.

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