Structural Differences in the Minimal Catalytic Domains of the GTPase-activating Proteins p120GAP and Neurofibromin

doi:10.1074/jbc.271.27.16409

Structural Differences in the Minimal Catalytic Domains of the GTPase-activating Proteins p120^GAP and Neurofibromin*

From the ^‡ Max-Planck-Institut für Molekulare Physiologie, Abteilung Strukturelle Biologie, Rheinlanddamm 201, 44139 Dortmund, Germany,
the ^§ Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie an der Universität Hamburg, Martinistraße 52, 20251 Hamburg, Germany, and
^¶ Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, 69009 Heidelberg, Germany

^∥ To whom correspondence should be addressed.

Abstract

The kinetic properties for the enzymatic stimulation of the GTPase reaction of p21^ras by the two GTPase-activating proteins (GAPs) p120^GAP and neurofibromin are different. In order to understand these differences and since crystallization attempts have only been successful with truncated fragments, structure/function requirements of the catalytic core of these proteins were investigated. Differences in size of the minimal catalytic domains of these two proteins were found as determined by limited proteolysis. The minimal catalytic domain has a molecular mass of 30 kDa in the case of p120^GAP and of 26 kDa in the case of neurofibromin. Both catalytic domains contain the homology boxes as well as the residues perfectly conserved among all Ras GAPs. The C termini of these fragments are identical, whereas the N-terminal part of the minimal p120^GAP domain is 47 amino acids longer. These newly identified minimal catalytic fragments were as active in stimulating GTPase activity toward p21^ras as the corresponding larger fragments GAP-334 and NF1-333 from which they had been generated via proteolytic digestion. Recently it was postulated that a fragment of 91 amino acids from neurofibromin located outside the conserved domain contains catalytic activity. In our hands this protein is unstable and has no catalytic activity. Thus, we believe that we have defined the true minimal domains of p120^GAP (GAP-273, residues Met⁷¹⁴-His⁹⁸⁶) and neurofibromin (NF1-230, residues Asp¹²⁴⁸-Phe¹⁴⁷⁷), which can be expressed via LMM fusion vectors in Escherichia coli and isolated in high purity.

Footnotes

↵* 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.
↵¹ The abbreviations used are:

GAP

GTPase-activating protein

IRA

inhibitory regulator of the RAS-cAMP pathway

LMM

light meromyosin

NF1

product of the neurofibromatosis type 1 gene

SH2 and SH3

Src homology domains 2 and 3, respectively

PCR

polymerase chain reaction

Gpp(NH)p

guanosine 5′-(β,γ-imido)triphosphate.
↵² M. R. Ahmadian, unpublished observations.
↵³ A. Lautwein, unpublished observations.
- Received December 28, 1995.
- Revision received April 15, 1996.