PMID- 19356586
OWN - NLM
STAT- MEDLINE
DCOM- 20090501
LR  - 20131121
IS  - 1089-8638 (Electronic)
IS  - 0022-2836 (Linking)
VI  - 387
IP  - 2
DP  - 2009 Mar 27
TI  - Characterisation of the nucleotide exchange factor ITSN1L: evidence for a kinetic 
      discrimination of GEF-stimulated nucleotide release from Cdc42.
PG  - 270-83
LID - 10.1016/j.jmb.2009.01.056 [doi]
AB  - Cdc42, a member of the Ras superfamily of small guanine nucleotide binding 
      proteins, plays an important role in regulating the actin cytoskeleton, 
      intracellular trafficking, and cell polarity. Its activation is controlled by 
      guanine nucleotide exchange factors (GEFs), which stimulate the dissociation of 
      bound guanosine-5'-diphosphate (GDP) to allow guanosine-5'-triphosphate (GTP) 
      binding. Here, we investigate the exchange factor activity of the Dbl-homology 
      domain containing constructs of the adaptor protein Intersectin1L (ITSN1L), which 
      is a specific GEF for Cdc42. A detailed kinetic characterisation comparing 
      ITSN1L-mediated nucleotide exchange on Cdc42 in its GTP- versus GDP-bound state 
      reveals a kinetic discrimination for GEF-stimulated dissociation of GTP: The 
      maximum acceleration of the intrinsic mGDP [2'/3'-O-(N-methyl-anthraniloyl)-GDP] 
      release from Cdc42 by ITSN1L is accelerated at least 68,000-fold, whereas the 
      exchange of mGTP [2'/3'-O-(N-methyl-anthraniloyl)-GTP] is stimulated only up to 
      6000-fold at the same GEF concentration. The selectivity in nucleotide exchange 
      kinetics for GDP over GTP is even more pronounced when a Cdc42 mutant, F28L, is 
      used, which is characterised by fast intrinsic dissociation of nucleotides. We 
      furthermore show that both GTP and Mg2+ ions are required for the interaction 
      with effectors. We suggest a novel model for selective nucleotide exchange 
      residing on a conformational change of Cdc42 upon binding of GTP, which enables 
      effector binding to the Cdc42.GTP complex but, at the same time, excludes 
      efficient modulation by the GEF. The higher exchange activity of ITSN1L towards 
      the GDP-bound conformation of Cdc42 could represent an evolutionary adaptation of 
      this GEF that ensures nucleotide exchange towards the formation of the 
      signalling-active GTP-bound form of Cdc42 and avoids dissociation of the active 
      complex.
FAU - Kintscher, Carsten
AU  - Kintscher C
AD  - Department of Biomolecular Mechanisms, Max-Planck-Institute for Medical Research, 
      Jahnstrasse 29, D-69120 Heidelberg, Germany.
FAU - Groemping, Yvonne
AU  - Groemping Y
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20090203
PL  - Netherlands
TA  - J Mol Biol
JT  - Journal of molecular biology
JID - 2985088R
RN  - 0 (Adaptor Proteins, Vesicular Transport)
RN  - 0 (Guanine Nucleotide Exchange Factors)
RN  - 0 (ITSN1 protein, human)
RN  - 0 (Mutant Proteins)
RN  - 146-91-8 (Guanosine Diphosphate)
RN  - 86-01-1 (Guanosine Triphosphate)
RN  - EC 3.6.5.2 (cdc42 GTP-Binding Protein)
RN  - I38ZP9992A (Magnesium)
SB  - IM
MH  - Adaptor Proteins, Vesicular Transport/chemistry/*metabolism
MH  - Amino Acid Substitution
MH  - Animals
MH  - Calorimetry
MH  - Cattle
MH  - Crystallography, X-Ray
MH  - Guanine Nucleotide Exchange Factors/*metabolism
MH  - Guanosine Diphosphate/metabolism
MH  - Guanosine Triphosphate/metabolism
MH  - Kinetics
MH  - Magnesium/metabolism
MH  - Models, Biological
MH  - Mutant Proteins/metabolism
MH  - Protein Structure, Tertiary
MH  - Titrimetry
MH  - cdc42 GTP-Binding Protein/*metabolism
EDAT- 2009/04/10 09:00
MHDA- 2009/05/02 09:00
CRDT- 2009/04/10 09:00
PHST- 2008/10/01 00:00 [received]
PHST- 2009/01/15 00:00 [revised]
PHST- 2009/01/26 00:00 [accepted]
PHST- 2009/04/10 09:00 [entrez]
PHST- 2009/04/10 09:00 [pubmed]
PHST- 2009/05/02 09:00 [medline]
AID - S0022-2836(09)00117-X [pii]
AID - 10.1016/j.jmb.2009.01.056 [doi]
PST - ppublish
SO  - J Mol Biol. 2009 Mar 27;387(2):270-83. doi: 10.1016/j.jmb.2009.01.056. Epub 2009 
      Feb 3.