Adhesion signaling: PAK meets Rac on solid ground
Interaction of cells with the extracellular matrix
influences various aspects of cellular behavior. A recent
study shows that cell–substrate adhesion is necessary
for effective coupling of the small GTPase Rac to its
Address: The Picower Institute for Medical Research, 350 Community
Drive, Manhasset, New York 11030, USA.
Current Biology 2000, 10:R535–R537
0960-9822/00/$ – see front matter
© 2000 Elsevier Science Ltd. All rights reserved.
Rac, a member of the Rho family of small GTP-binding
proteins, mediates the biological effects of a wide range of
growth factors, cytokines and adhesion molecules, thereby
regulating the organization of the actin cytoskeleton, cell
migration, cell proliferation, vesicle trafficking and gene
transcription [1,2]. Integrin signaling plays an important
role in several Rac-regulated functions, such as cell motil-
ity  and cell cycle control . In both of these
processes, the integration of signals from growth factors
and integrins is thought to have a critical role. It has
remained unclear whether the interaction of cells with
extracellular matrix proteins leads to Rac activation and
whether growth-factor-stimulated signaling through Rac is
modulated by cell attachment to the extracellular matrix.
These issues have now been addressed in a recent paper
by del Pozo et al. , who have shown that in fibroblasts
serum growth factors and cell adhesion to the extracellular
matrix both contribute to Rac activation. Interestingly, del
Pozo et al. observe that cell–substrate adhesion also
strongly stimulates the coupling of activated Rac to the
serine/threonine kinase PAK, a downstream effector of
Rac, revealing a novel mode of regulating Rac-controlled
Control of Rac activation
Most members of the Ras and Rho GTPase families cycle
between the GDP-bound (inactive) state and GTP-bound
(active) state. The nucleotide state of Rho family proteins
is controlled by three classes of regulatory proteins .
Guanine nucleotide exchange factors (GEFs) catalyze the
exchange of GDP for GTP. GTPase-activating proteins
(GAPs) promote the intrinsic GTP hydrolysis by Rho
GTPases, leading to their rapid conversion to the GDP-
bound state, whereas Rho guanine nucleotide dissociation
inhibitors (RhoGDIs) preferentially bind to the GDP-
bound form of Rho proteins and prevent both spontaneous
and GEF-catalyzed release of nucleotide. RhoGDIs there-
fore appear to maintain Rho proteins in the inactive state.
As is the case for Ras family members, Rho GTPases are
post-translationally modified by attachment of an iso-
prenoid lipid group, which serves to anchor these GTPases
to membrane compartments. RhoGDIs preferentially bind
to lipid-modified Rho proteins, and inhibit their interac-
tion with cellular membranes.
Adhesion control of Rac—PAK coupling
Growth-factor-induced activation of many signaling path-
ways is shut down upon detachment of cells from the
extracellular matrix [7,8]. Somewhat surprisingly then, del
Pozo et al.  found that serum leads to a significant
increase in the level of active Rac in suspended fibro-
blasts. Nucleotide exchange on Rac is thought to occur at
the plasma membrane . Activation of Rac in suspension
conditions, however, does not seem to be accompanied by
translocation of Rac to the plasma membrane, raising the
possibility that GEFs may be able to interact productively
with Rac in the cytosol.
The p21-activated kinase (PAK) family comprises the best
characterized effectors of Rac and Cdc42 to date and is
thought to mediate cytoskeletal reorganization and tran-
scription downstream of these GTPases . The kinase
activity of PAK is strongly stimulated by binding to acti-
vated Rac or Cdc42, both in vitro and in vivo. PAK is also
the main Rac effector for which changes in activity in
response to physiological stimuli can be readily deter-
mined. del Pozo et al.  noted that, whereas the level of
activated Rac in serum-stimulated cells in suspension
reaches almost half of that in serum-stimulated attached
cells, in suspension conditions this pool of activated Rac is
unable to stimulate PAK activity, although serum strongly
stimulates PAK in adherent cells. These results suggest
that, in addition to increasing Rac–GTP levels, attach-
ment to extracellular matrix also enhances the ability of
activated Rac to stimulate PAK.
Earlier studies by Mayer and colleagues  had indicated
that localization of PAK to the plasma membrane may
have an important role in the activation of PAK in vivo.
The adaptor protein Nck is a good candidate for mediating
the interaction of PAK with the membrane . Data
obtained by del Pozo et al.  also support the notion that
membrane localization is essential for PAK activation. This
group showed that expression of a constitutively active
mutant of Rac that lacks a membrane-targeting sequence
fails to activate PAK in adherent cells. Moreover, low-level
expression of an activated mutant of Rac that is constitu-
tively targeted to the membrane — either via inclusion of
a myristylation sequence or by fusion to a heterologous
transmembrane domain — leads to high levels of activa-
tion of PAK in suspension cultures.
Models for the regulation of Rac–PAK coupling
It is intriguing that del Pozo et al.  observe only rela-
tively minor increases in Rac activation and membrane
translocation upon serum stimulation of adherent cells, but
that these changes lead to strong enhancement of PAK
activity. In addition, whereas attachment to the extracellu-
lar matrix causes a significant increase in PAK localization
to the membrane, serum stimulation of adherent cells does
not cause additional PAK translocation to the membrane.
These results suggest that Rac membrane localization is
the critical factor in serum-induced PAK activation.
One potential mechanism for the increased coupling of
activated Rac to PAK is that in adherent cells serum
induces translocation of Rac to a membrane microdomain.
This process in turn could lead to an increase in the local
concentration of Rac and concomitant stimulation of
Rac–PAK complex formation . In support of an
important role for the targeting of Rac to the plasma
membrane in the regulation of Rac signaling, del Pozo
et al. found that recombinant constitutively active Rac
present in the cytosol of suspension cells efficiently binds
to membranes prepared from adherent cells, but not to
membranes prepared from suspended cells. Rafts and
caveoli, membrane domains that are rich in glycosphin-
golipids and cholesterol , would be good candidates
for membrane compartments that aid in the recruitment
of Rac. This proposal is supported by recent data showing
that platelet-derived growth factor stimulates redistribu-
tion of Rac to caveoli . Thus, the increased binding of
Rac to plasma membranes prepared from attached cells
compared with membranes from suspension cells might
be because rafts are absent from suspension cells
The molecular mechanisms that regulate the binding of
GTPases to membranes remain to be clarified. As men-
tioned earlier, RhoGDIs are thought to play a major role in
the regulation of the GTPase–membrane interaction .
It would therefore be interesting to determine whether
plasma membrane preparations from attached cells
contain factors that stimulate the release of RhoGDI from
Rac. A potential role for RhoGEFs in membrane targeting
of Rac by adhesion should also be considered. Growth
factors have been shown to induce the translocation of the
exchange factors Vav2 and Tiam1 to the membrane com-
partment [16,17]. The data obtained by del Pozo et al. 
clearly show, however, that regulated membrane binding
of Rac can occur independently of nucleotide exchange,
so the role of RhoGEFs in membrane targeting of Rac
remains to be clarified.
The GTPase ADP-ribosylation factor 6 (ARF6) has also
been implicated in the regulation of Rac recruitment to the
plasma membrane. ARF6 is thought to control endosome
recycling to the plasma membrane . Stimulation of cells
with the G-protein-coupled receptor agonist bombesin has
Current Biology Vol 10 No 14
(a) Suspension cell – serum
Rac–GDP Rac–GTPRhoGDI Inactive PAKActive PAK
(b) Adherent cell – serum(c) Adherent cell + serum
Coupling of PAK to activated Rac is mediated by membrane
recruitment of Rac in adherent cells. The activation and distribution of
Rac and PAK in (a) a suspension cell in the absence of serum, (b) an
adherent cell in the absence of serum and (c) an adherent cell in the
presence of serum. Cell attachment in the absence of serum induces a
slight increase in Rac activation and plasma membrane recruitment
compared with that seen in serum-free suspension cells. PAK also
redistributes to the membrane, and is partially activated. Serum
stimulation of adherent cells causes further recruitment of Rac to the
plasma membrane. Redistribution of activated Rac (and possibly PAK)
to membrane microdomains (shown as darker shading in the
membrane in (c)) might increase the local concentration of activated
Rac and enhance complex formation with PAK.
recently been shown to cause ARF6-dependent redistribu-
tion of ARF6- and Rac1-containing vesicles to the cell
surface and concomitant lamellipodia formation .
Although the precise relationship between Rac and ARF6
remains controversial, this and similar observations  raise
the possibility that ARF6 might also mediate adhesion-stim-
ulated redistribution of Rac to the plasma membrane.
Implications for cell physiology
The modulation of Rac-controlled signaling pathways by
cell–substrate adhesion, as highlighted by the work of del
Pozo et al. , may be important for a large number of
physiological processes that are known to be affected by
the interaction of cells with the extracellular matrix. This
regulation could contribute to anchorage-dependent cell
proliferation and may constitute a control element in
angiogenesis, morphogenetic movements during develop-
ment and other biological functions that involve directed
cell movement. Further elucidation of this control mecha-
nism may therefore shed new light on these events. It will
be of interest to determine whether such a mechanism
holds for the coupling of Rac to additional effectors and
whether signaling pathways controlled by other Rho
family members are modulated by adhesion signaling in a
I thank Sal Coniglio, Crislyn D’Souza-Schorey, Kirk Manogue and Maria
Ruggieri for helpful comments on the manuscript. I am supported by the
National Institutes of Health grant CA-87567-01 and a grant from the
1. Hall A: Rho GTPases and the actin cytoskeleton. Science 1998,
2. Van Aelst L, D’Souza-Schorey C: Rho GTPases and signaling
networks. Genes Dev 1997, 11:2295-2322.
3. Lauffenburger DA, Horwitz AF: Cell migration: a physically
integrated molecular process. Cell 1996, 84:359-369.
4. Giancotti FG, Ruoslahti E: Integrin signaling. Science 1999,
5. del Pozo MA, Price LS, Alderson NB, Ren XD, Schwartz MA:
Adhesion to the extracellular matrix regulates the coupling of
the small GTPase Rac to its effector PAK. EMBO J 2000,
6. Boguski MS, McCormick F: Proteins regulating Ras and its
relatives. Nature 1993, 366:643-654.
7. Renshaw MW, Ren XD, Schwartz MA: Growth factor activation of
MAP kinase requires cell adhesion. EMBO J 1997, 16:5592-5599.
8. Le Gall M, Chambard JC, Breittmayer JP, Grall D, Pouyssegur J, Van
Obberghen-Schilling E: The p42/p44 MAP kinase pathway
prevents apoptosis induced by anchorage and serum removal.
Mol Biol Cell 2000, 11:1103-1112.
9. Bokoch GM, Bohl BP, Chuang TH: Guanine nucleotide exchange
regulates membrane translocation of Rac/Rho GTP-binding
proteins. J Biol Chem 1994, 269:31674-31679.
10. Manser E, Lim L: Roles of PAK family kinases. Prog Mol Subcell
Biol 1999, 22:115-133.
11. Lu W, Katz S, Gupta R, Mayer BJ: Activation of Pak by membrane
localization mediated by an SH3 domain from the adaptor protein
Nck. Curr Biol 1997, 7:85-94.
12. Kholodenko BN, Hoek JB, Westerhoff HV: Why cytoplasmic
signalling proteins should be recruited to cell membranes. Trends
Cell Biol 2000, 10:173-178.
13. Kurzchalia TV, Parton RG: Membrane microdomains and caveolae.
Curr Opin Cell Biol 1999, 11:424-431.
14. Michaely PA, Mineo C, Ying YS, Anderson RG: Polarized
distribution of endogenous Rac1 and RhoA at the cell surface.
J Biol Chem 1999, 274:21430-21436.
15. Olofsson B: Rho guanine dissociation inhibitors: pivotal molecules
in cellular signalling. Cell Signal 1999, 11:545-554.
16. Pandey A, Podtelejnikov AV, Blagoev B, Bustelo XR, Mann M, Lodish
HF: Analysis of receptor signaling pathways by mass
spectrometry: identification of vav-2 as a substrate of the
epidermal and platelet-derived growth factor receptors. Proc Natl
Acad Sci USA 2000, 97:179-184.
17. Buchanan FG, Elliot CM, Gibbs M, Exton JH: Translocation of the
rac1 guanine nucleotide exchange factor tiam1 induced by
platelet-derived growth factor and lysophosphatidic acid. J Biol
Chem 2000, 275:9742-9748.
18. D’Souza-Schorey C, van Donselaar E, Hsu VW, Yang C, Stahl PD,
Peters PJ: ARF6 targets recycling vesicles to the plasma
membrane: insights from an ultrastructural investigation. J Cell
Biol 1998, 140:603-616.
19. Boshans RL, Szanto S, Van Aelst L, D’Souza-Schorey C:
ADP-ribosylation factor 6 regulates actin cytoskeleton remodeling
in coordination with Rac1 and RhoA. Mol Cell Biol 2000,
20. Radhakrishna H, Al-Awar O, Khachikian Z, Donaldson JG: ARF6
requirement for Rac ruffling suggests a role for membrane
trafficking in cortical actin rearrangements. J Cell Sci 1999,