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J. Biol. Chem., Vol. 278, Issue 8, 5894-5901, February 21, 2003
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From the The GTPase dynamin-2 (dyn-2)
binds and positively regulates the nitric oxide-generating enzyme,
endothelial nitric-oxide synthase (eNOS) (Cao, S., Yao, Y.,
McCabe, T., Yao, Q., Katusic, Z., Sessa, W., and Shah, V. (2001) J. Biol. Chem. 276, 14249-14256). Here we
demonstrate, using purified proteins, that this occurs through a
selective influence of the dyn-2 proline-rich domain (dyn-2 PRD) on the
eNOS reductase domain. In vitro studies demonstrate that
dyn-2 PRD fused with glutathione S-transferase (GST) binds recombinant eNOS protein specifically and with binding kinetics comparable with that observed between dyn-2 full-length and eNOS. Additionally, GST-dyn-2 PRD binds the in vitro transcribed
35S-eNOS reductase domain but not the 35S-eNOS
oxygenase domain. Furthermore GST-dyn-2 PRD binds a
35S-labeled eNOS reductase domain fragment (amino acids
645-850) that partially overlaps with the FAD binding domain of eNOS.
A recombinant form of the SH3-containing protein Fyn competes the binding of recombinant eNOS protein with dyn-2 PRD, thereby implicating the SH3-like region contained within this reductase domain fragment as
the dyn-2 binding region. Mammalian two-hybrid screen corroborates these interactions in cells as well. Functional studies demonstrate that dyn-2 PRD selectively potentiates eNOS activity in a
concentration-dependent manner in an order of magnitude
similar to that observed with dyn-2 full-length and in a manner that
requires calmodulin. Although dyn-2 PRD does not influence eNOS
oxygenase domain function or ferricyanide reduction, it does potentiate
the ability of recombinant eNOS to reduce cytochrome c,
supporting an influence of dyn-2 PRD on electron transfer between FAD
and FMN. (These data indicate that the binding domains of dyn-2 and
eNOS reside within the dyn-2 PRD domain and the FAD binding region of
the eNOS reductase domains, respectively, and that dyn-2 PRD is
sufficient to mediate dyn-2-dependent potentiation of eNOS
activity, at least in part, by potentiating electron transfer.)
The Proline-rich Domain of Dynamin-2 Is Responsible for
Dynamin-dependent in Vitro Potentiation of
Endothelial Nitric-oxide Synthase Activity via Selective Effects
on Reductase Domain Function*
,, and§¶
Gastrointestinal Research Unit and
Department of Medicine and the § Tumor Biology Program,
Mayo Clinic, Rochester, Minnesota 55905
*
This work was supported by National Institutes of Health
Grants DK-02529, DK-59998, and DK-59615 (to V. S.), a grant from the
Northland Affiliate of the American Heart Association (to V. S.), a
fellowship grant from the Northland Affiliate of the American Heart
Association (to S. C.), and by the Mayo Clinic Foundation.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
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