Regulation of Class IA PI 3-kinases: C2 domain-iSH2
domain contacts inhibit p85/p110? and are disrupted
in oncogenic p85 mutants
Haiyan Wua, S. Chandra Shekara, Rory J. Flinna, Mirvat El-Sibaib, Bijay S. Jaiswalc, K. Ilker Send,
Vasantharajan Janakiramanc, Somasekar Seshagiric, Gary J. Gerfend, Mark E. Girvine, and Jonathan M. Backera,1
aDepartments of Molecular Pharmacology,dPhysiology and Biophysics, andeBiochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue,
Bronx, NY, 10461;bDepartment of Natural Sciences, Lebanese American University, Beirut Campus, Beirut, Lebanon; andcDepartment of Molecular Biology,
Genetech Inc., 1 DNA Way, South San Francisco, CA 94080
Edited by Peter K. Vogt, Scripps Research Institute, La Jolla, CA, and approved October 7, 2009 (received for review March 3, 2009)
We previously proposed a model of Class IA PI3K regulation in
which p85 inhibition of p110? requires (i) an inhibitory contact
between the p85 nSH2 domain and the p110? helical domain, and
(ii) a contact between the p85 nSH2 and iSH2 domains that orients
the nSH2 so as to inhibit p110?. We proposed that oncogenic
contact. However, we now find that within the context of a
minimal regulatory fragment of p85 (the nSH2-iSH2 fragment,
termed p85ni), the nSH2 domain rotates much more freely
domain. These data are not compatible with our previous model.
We therefore tested an alternative model in which oncogenic p85
truncations destabilize an interface between the p110? C2 domain
(residue N345) and the p85 iSH2 domain (residues D560 and N564).
p85ni-D560K/N564K shows reduced inhibition of p110?, similar to
the truncated p85ni-572STOP. Conversely, wild-type p85ni poorly
inhibits p110?N345K. Strikingly, the p110?N345K mutant is inhib-
ited to the same extent by the wild-type or truncated p85ni,
suggesting that mutation of p110?-N345 is not additive with the
p85ni-572STOPmutation. Similarly, the D560K/N564K mutation is
not additive with the p85ni-572STOPmutant for downstream sig-
naling or cellular transformation. Thus, our data suggests that
mutations at the C2-iSH2 domain contact and truncations of the
iSH2 domain, which are found in human tumors, both act by
disrupting the C2-iSH2 domain interface.
cancer ? glioblastoma ? phosphoinositide 3-kinase ? PIK3CA
naling contributes to cancer and other human diseases (1). Class
IA PI 3-kinases, which produce PI[3,4,5]P3 in intact cells (2), are
p50?, or p55?) and a catalytic subunit (p110?, p110?, or p110?)
(reviewed in ref. 3). The regulatory subunits have two major
functions: they stabilize the catalytic subunits against thermal
denaturation, and they maintain the catalytic subunit in an
inhibited, low activity state (4, 5).
p85 and p110 are both multidomain proteins that bind to each
other and to upstream activators such as Rac and Cdc42, Ras,
and tyrosine phosphorylated receptors and adapters (reviewed
in ref. 6). p85 contains an SH3 domain, a Rac/Cdc42-binding
domain homologous to a GAP domain in the BCR gene product,
and two SH2 domains that flank an antiparallel coiled coil
domain (the iSH2 domain). While NMR, EPR, and crystal
structures have been obtained for the individual domains (7–15),
there are currently no structures that define how these domains
are arranged in space. The p110? catalytic subunit has been
domain (ABD) or the entire p110? bound to the coiled coil
(iSH2) domain of p85 (15, 16). Like the related Class IB catalytic
subunit p110? (17), p110? contains Ras-binding, C2, helical, and
I 3-kinases are important cellular regulators of growth,
survival, and motility, and deregulation of PI 3-kinase sig-
binding of the N-terminal ABD to the far end of the rod-like
iSH2 domain, consistent with previous biochemical studies
(18–20). The kinase and C2 domains drape over the iSH2
domain like a saddle, with the Ras-binding domain facing
upward above the ABD. The helical domain is positioned at the
opposite end of the molecule from the ABD, and is therefore
close to the ends of the iSH2 domain that are linked to the two
Structural studies on p110? and p110? have not provided a
mechanism to explain the inhibition of p110? by p85 binding, or
the activation of p85/p110? dimers by phosphoprotein binding
to the SH2 domains of p85 (21, 22). We and others have shown
that the iSH2 domain-ABD interface is structurally rigid and
does not regulate p110 activity (23–25). In contrast, the N-
terminal SH2 (nSH2) domain of p85 is required for inhibition of
p110?. Recent biochemical studies suggest that basic residues
surrounding the phosphopeptide binding site in the nSH2 do-
main make an inhibitory contact with an acidic patch in the
helical domain of p110? (15). Phosphoprotein binding to the
SH2 domain would presumably disrupt this inhibitory contact
and activate the p85/p110 dimer. This interface is also disrupted
by oncogenic mutations in the helical domain of p110 that have
been identified in human cancers (15, 26). Helical domain
mutations in p110? synergize with activated Ras for activation of
PI 3-kinase (27).
Oncogenic mutations have been described in p85, mostly
truncations or deletions in the C-terminal end of the iSH2
domain (28–30); more recent sequencing studies have identified
additional deletion and point mutations in the iSH2 domain (31,
32). The oncogenic p85 mutations presumably act by disrupting
inhibitory contacts with p110?, and the p85572STOPand p85
(?583–605) mutants (28, 30) fail to inhibit p110? in vitro (33).
We previously examined the structure of the minimal regulatory
portion of p85, the nSH2-iSH2 fragment (p85ni) (23). Based on
amide protons by spin probes in the iSH2 domain within p85ni,
we proposed that contacts between the C-terminal end of the
iSH2 domain and the nSH2 domain position the latter domain
such that it forms an inhibitory contact with p110? (33). Loss of
these contacts in the oncogenic p85 mutants would explain the
loss of p110? inhibition. However, several recent data have led
M.E.-S., and B.S.J. performed research; V.J. and S.S. contributed new reagents/analytic
tools; H.W., S.C.S., M.E.-S., K.I.S., G.J.G., M.E.G., and J.M.B. analyzed data; and M.E.G. and
J.M.B. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
December 1, 2009 ?
vol. 106 ?
Transformation Assays. NIH 3T3 cells were transiently transfected with myc-
p110? and wild-type or mutant p85ni. Two days after transfection, the cells
agar, in a six-well dish. Cell colonies were counted after 3 weeks.
Anti-PIP3 Staining. Cells were fixed and stained with anti-HA antibody (to
fluorescence quantification, all digital images were imported in NIH image
software and analyzed by using a previously described macro (45). This macro
collects pixel intensities from the perimeter of the cell in a 0.22-m stepwise
manner. The pixel intensities in the leading-edge compartment, defined as
0.66 m from the perimeter of the cells, were averaged and normalized to the
edge intensity of nonstimulated cells.
ACKNOWLEDGMENTS. This work was supported by the Janey Fund and the
National Institutes of Health Grants GM55692 (to J.M.B.), GM072085 (to
M.E.G.), and GM075920 (to G.J.G.), the Albert Einstein Comprehensive Cancer
DK020541. J.M.B and M.E.G are members of the New York Structural Biology
Center (NYSBC). The NYSBC is a Strategically Targeted Research Center sup-
ported by the New York State Office of Science, Technology, and Academic
Research. NYSBC NMR resources are supported by the National Institutes of
Health Grant P41 GM66354.
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