Abstract: | Integrated cascades of protein tyrosine and serine/threonine
phosphorylation play essential roles in transducing signals in response to
growth factors and cytokines. How adaptor or scaffold proteins assemble
signaling complexes through both phosphotyrosine and phosphoserine/threonine
residues to regulate specific signaling pathways and biological responses is
unclear. We show in multiple cell types that endogenous 14-3-3ζ is
phosphorylated on Tyr179 in response to granulocyte macrophage
colony-stimulating factor. Importantly, 14-3-3ζ can function as an
intermolecular bridge that couples to phosphoserine residues and also directly
binds the SH2 domain of Shc via Tyr179. The assembly of these
14-3-3:Shc scaffolds is specifically required for the recruitment of a
phosphatidylinositol 3-kinase signaling complex and the regulation of CTL-EN
cell survival in response to cytokine. The biological significance of these
findings was further demonstrated using primary bone marrow-derived mast cells
from 14-3-3ζ-/- mice. We show that cytokine was able to
promote Akt phosphorylation and viability of primary mast cells derived from
14-3-3ζ-/- mice when reconstituted with wild type
14-3-3ζ, but the Akt phosphorylation and survival response was reduced in
cells reconstituted with the Y179F mutant. Together, these results show that
14-3-3:Shc scaffolds can act as multivalent signaling nodes for the
integration of both phosphoserine/threonine and phosphotyrosine pathways to
regulate specific cellular responses.The ability of a cell to respond to extrinsic stimuli critically hinges on
its ability to regulate specific intracellular protein-protein interactions in
a reversible manner. Such signals are relayed within the cell through the
assembly of signaling complexes that are built using protein scaffolds. One
important mechanism by which this occurs is via the binding of Src homology 2
(SH2)5 or
phosphotyrosine-binding (PTB) domains to phosphotyrosine residues
(1,
2). Importantly, the ability of
individual SH2 or PTB domains to recognize specific phosphotyrosine motifs in
different proteins enables the assembly of purpose-built signaling complexes
that promote signaling via specific pathways
(3). In some cases, signaling
proteins not only contain more than one SH2 and/or PTB domain but are also
themselves tyrosine-phosphorylated, leading to a network of
phosphotyrosine-mediated protein-protein interactions.Although less well studied, phosphoserine/threonine-binding proteins are
also important for the assembly of signaling complexes. For example, the
14-3-3 family of proteins is able to bind phosphoserine/threonine residues in
a sequence-specific context (RSX(S/T)XP and
RXXX(S/T)XP, where (S/T) is phosphoserine/threonine)
(4,
5). The 14-3-3 proteins have
been proposed to function as “modifiers” or
“sequestrators”; however, because of their dimeric structure, they
have also been proposed to function as “adaptor” or
“scaffold” proteins through their ability to bring together two
serine/threonine phosphorylated proteins
(4–7).
Additionally, a number of phosphoserine/threonine-binding modules such as
tryptophan-tryptophan (WW), Forkhead-associated (FHA), Polo box (PBD), and
BRCA1 C-terminal (BRCT) domains have been shown to interact with
phosphoserine/threonine residues within a sequence-specific context and have
also been proposed to be important for the assembly of multi-protein signaling
complexes (8).The genes/cassettes encoding each phosphotyrosine- and
phosphoserine/threonine-binding protein/module arose as a separate
evolutionary event, and the DNA encoding these modules has been subject to
frequent duplication and shuffling. For example, the 14-3-3 family of proteins
is ubiquitously expressed in mammalian tissues and is composed of seven
different isoforms, each encoded by a separate gene
(6). In addition, duplication
and shuffling of SH2, PTB, WW, FHA, PBD, and BCRT cassettes has led to their
wide distribution among signaling proteins. Yet, despite the frequent
duplication and shuffling of the DNA encoding these domains throughout
evolution, proteins that contain both a phosphotyrosine-binding cassette
(e.g. SH2 or PTB) and a phosphoserine/threonine-binding cassette
(e.g. 14-3-3, WW, FHA, PBD, and BCRT) have not been identified. This
is perhaps surprising given the highly integrated nature of phosphotyrosine
and phosphoserine/threonine signaling and would suggest that alternative
strategies to regulate integration are at play.We show here that 14-3-3ζ is tyrosine-phosphorylated, enabling it to
interact with Shc and provide a scaffold for the assembly of signaling
complexes via both phosphoserine/threonine and phosphotyrosine residues. Our
results show that Tyr179 of 14-3-3ζ directly binds to the SH2
domain of Shc and that this interaction is critical for the assembly of a
phosphatidylinositol (PI) 3-kinase signaling complex in response to
granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation.
Moreover, we show that Tyr179 of 14-3-3ζ is necessary and
sufficient for the ability of GM-CSF to regulate PI 3-kinase and cell survival
in the CTL-EN line. Furthermore, reconstitution of primary mast cells derived
from 14-3-3ζ-/- mice with wild type (wt) or mutant
14-3-3ζ demonstrated an important role for Tyr179 in
cytokine-mediated Akt phosphorylation and cell survival. These multivalent
14-3-3:Shc scaffolds provide a novel mechanism by which
phosphoserine/threonine and phosphotyrosine pathways can be integrated for the
regulation of specific cellular responses. |