LETTERS
Rewiring cellular morphology pathways with
synthetic guanine nucleotide exchange factors
Brian J. Yeh1,2*, Robert J. Rutigliano3*, Anrica Deb2, Dafna Bar-Sagi3,4 & Wendell A. Lim2
Eukaryotic cells mobilize the actin cytoskeleton to generate a
remarkable diversity of morphological behaviours, including
motility, phagocytosis and cytokinesis. Much of this diversity is
mediated by guanine nucleotide exchange factors (GEFs) that
activate Rho family GTPases—the master regulators of the actin
cytoskeleton1–3. There are over 80 Rho GEFs in the human genome
(compared to only 22 genes for the Rho GTPases themselves), and
the evolution of new and diverse GEFs is thought to provide a
mechanism for linking the core cytoskeletal machinery to a wide
range of new control inputs. Here we test this hypothesis and ask
if we can systematically reprogramme cellular morphology by
engineering synthetic GEF proteins. We focused on Dbl family
Rho GEFs, which have a highly modular structure common to
many signalling proteins4,5: they contain a catalytic Dbl homology
(DH) domain linked to diverse regulatory domains,many ofwhich
autoinhibit GEF activity2,3. Here we show that by recombining
catalytic GEF domains with new regulatory modules, we can gen-
erate synthetic GEFs that are activated by non-native inputs. We
have used these synthetic GEFs to reprogramme cellular behaviour
in diverse ways. The GEFs can be used to link specific cytoskeletal
responses to normally unrelated upstream signalling pathways. In
addition, multiple synthetic GEFs can be linked as components in
series to form an artificial cascade with improved signal proces-
sing behaviour. These results show the high degree of evolutionary
plasticity of this important family of modular signalling proteins,
and indicate that it may be possible to use synthetic biology
approaches to manipulate the complex spatio-temporal control
of cell morphology.
Rho family GTPases are central signalling molecules in the regu-
lation of the actin cytoskeleton1 (Fig. 1a). These proteins are con-
formational switches that exist in GDP- and GTP-bound states;
however, only the GTP-bound state actively transduces signal to
downstream effectors. Cycling between states is primarily controlled
by opposing enzymes: GTPase activating proteins promote hydro-
lysis of bound GTP to GDP (inactivation), whereas GEFs promote
exchange of bound GDP for GTP (activation). The three canonical
members of the Rho family—Cdc42, Rac1 and RhoA—stimulate the
distinct morphological outputs of protrusive filopodia (thin actin
microspikes), protrusive lamellipodia (broad membrane ruffles)
and contractile actin:myosin filaments, respectively.
As an initial target for rewiring GTPase signalling, we attempted
to reprogramme Dbl family GEFs (Fig. 1b) so that their activity
was controlled by protein kinase A (PKA), a well-characterized
prototypical kinase6 (Fig. 2a). We first designed a PKA-sensitive
autoinhibitory module, inspired by natural examples7, that consisted
of a PDZ (PSD95, Dlg, ZO-1) domain–peptide interaction pair that
could be disrupted by PKA phosphorylation. The syntrophin PDZ
domain recognizes short carboxy-terminal peptide motifs (con-
sensus sequence (R/K)E(S/T)xy-COOH; y denotes aliphatic resi-
dues)8, which are close in sequence to the ideal PKA substrate
(RRRRSIIFI)9. A hybrid sequence (RRRESIV-COOH) could serve
both as an interaction ligand for the syntrophin PDZ domain and
*These authors contributed equally to this work.
1Chemistry and Chemical Biology Graduate Program, University of California, San Francisco, San Francisco, California 94158-2517, USA. 2Department of Cellular and Molecular
Pharmacology and the Cell Propulsion Lab, UCSF/UCB NIH Nanomedicine Development Center, University of California, San Francisco, San Francisco, California 94158-2517, USA.
3Department of Molecular Genetics and Microbiology, School of Medicine, State University of New York at Stony Brook, Stony Brook, New York 11794, USA. 4Department of
Biochemistry, New York University School of Medicine, New York, New York 10016, USA.
GTPase
GEFs
a b c
Natural Novel
x
DH-PH
DH-PH
DH-PH
a
b
x
Autoinhibitory
domain(s)
Catalytic
GEF domain
Input
DH-PH
+
a
b
PathogenInputs
Natural GEFs
Synthetic GEF
Morphological
output
CB D XA
+
−
GDP GTP
Figure 1 | GEFs link diverse inputs to Rho GTPasemodules that control cell
morphology. a, GEFs functionally connect signalling inputs to activation of
Rho GTPases, which regulate morphology of the actin cytoskeleton. Some
bacterial pathogens encode GEFs that activate host GTPases30. Synthetic
GEFs could, in principle, mediate new connections in living cells. b, The
largest family of Rho GEFs are Dbl-related proteins, which share a catalytic
DH-PH core. In many cases, adjacent modular domains mediate
autoinhibitory interactions that can be disrupted by specific inputs. Here we
exploit this modular structure to construct synthetic GEFs.
Vol 447 |31 May 2007 |doi:10.1038/nature05851
596
Nature ©2007 Publishing Group