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Lipid and Protein Co-Regulation of PI3K Effectors Akt and Itk in Lymphocytes

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The phosphoinositide 3-kinase (PI 3-kinase, PI3K) pathway transduces signals critical for lymphocyte function. PI3K generates the phospholipid PIP3 at the plasma membrane to recruit proteins that contain pleckstrin homology (PH) domains - a conserved domain found in hundreds of mammalian proteins. PH domain-PIP3 interactions allow for rapid signal propagation and confer a spatial component to these signals. The kinases Akt and Itk are key PI3K effectors that bind PIP3 via their PH domains and mediate vital processes - such as survival, activation, and differentiation - in lymphocytes. Here, we review the roles and regulation of PI3K signaling in lymphocytes with a specific emphasis on Akt and Itk. We also discuss these and other PH domain-containing proteins as they relate more broadly to immune cell function. Finally, we highlight the emerging view of PH domains as multifunctional protein domains that often bind both lipid and protein substrates to exert their effects.
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REVIEW ARTICLE
published: 13 March 2015
doi: 10.3389/fimmu.2015.00117
Lipid and protein co-regulation of PI3K effectors Akt and Itk
in lymphocytes
Xinxin Wang1, Leonard Benjamin Hills2andYina Hsing Huang2,3*
1California Institute for Biomedical Research, La Jolla, CA, USA
2Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Lebanon, NH, USA
3Department of Pathology, Geisel School of Medicine at Dartmouth, Lebanon, NH, USA
Edited by:
Klaus Okkenhaug, Babraham
Institute, UK
Reviewed by:
M. Suresh, University of Wisconsin
Madison, USA
Leslie J. Berg, University of
Massachusetts Medical School, USA
Cosima T. Baldari, University of Siena,
Italy
*Correspondence:
Yina Hsing Huang, Departments of
Pathology and Microbiology and
Immunology,The Geisel School of
Medicine at Dartmouth, HB 7600,
Borwell 650E, One Medical Center
Drive, Lebanon, NH 03756, USA
e-mail: yina.h.huang@dartmouth.edu
The phosphoinositide 3-kinase (PI 3-kinase, PI3K) pathway transduces signals critical for
lymphocyte function. PI3K generates the phospholipid PIP3at the plasma membrane to
recruit proteins that contain pleckstrin homology (PH) domains – a conserved domain found
in hundreds of mammalian proteins. PH domain–PIP3interactions allow for rapid signal
propagation and confer a spatial component to these signals. The kinases Akt and Itk are
key PI3K effectors that bind PIP3via their PH domains and mediate vital processes – such
as survival, activation, and differentiation – in lymphocytes. Here, we review the roles and
regulation of PI3K signaling in lymphocytes with a specific emphasis on Akt and Itk. We
also discuss these and other PH domain-containing proteins as they relate more broadly
to immune cell function. Finally, we highlight the emerging view of PH domains as multi-
functional protein domains that often bind both lipid and protein substrates to exert their
effects.
Keywords: PI3K, lymphocyte activation, pleckstrin homology domain, Akt signaling, Itk signaling
LYMPHOCYTE ACTIVATION RECEPTORS SIGNAL THROUGH
CLASS I PI3Ks
Phosphoinositide 3-kinase (PI3K) activation is important for
lymphocyte survival, activation, differentiation, and migration.
Many lymphocyte surface receptors activate class 1 PI3Ks, which
phosphorylate phosphatidyl inositol 4,5-bisphosphate [PI(4,5)P2,
PIP2] at the D-3 hydroxyl group of the myo-inositol ring to gener-
ate phosphatidyl inositol 3,4,5-trisphosphate [PI(3,4,5)P3, PIP3].
Two subclasses, 1A and 1B, are activated by distinct receptor types
(Figure 1). Receptors or signaling adapters that are phosphory-
lated at YxxM sequence motifs signal though class IA PI3K, which
includes p85αand p85βregulatory subunits and p110α, p110β,
and p110δcatalytic subunits. These receptors include CD19,
CD28, and ICOS co-receptors; IL-2, IL-7, IL-3, IL-15, and GM-
CSF cytokine receptors (16); and receptors coupled to TRIM,
DAP10, and MyD88 adapter proteins (711). Receptor ligation
leads to tyrosine phosphorylation at the YxxM motif and sub-
sequent recruitment of PI3K regulatory subunits through one or
both Src homology 2 (SH2) domains. Regulatory subunits are then
phosphorylated by Syk or Jak family tyrosine kinases to trigger
activation of their constitutively associated catalytic subunits (3).
G-protein-coupled receptors (GPCRs) signal through Class 1B
PI3K, which includes p101 regulatory and p110γcatalytic sub-
units (12). These classic, seven transmembrane domain receptors
include chemokine receptors and signal through heterotrimeric
G proteins, Gαand Gβγ to promote cell migration. GPCR lig-
ation dissociates the Gβγ dimer, allowing its binding to p101
regulatory subunits and subsequent activation of associated p110γ
catalytic subunits. Activation of p110γcatalytic activity can also
be induced by Ras activation (Ras-GTP) to promote migration of
neutrophils (13).
Although many receptors activate class 1 PI3K, the magnitude
and kinetics of PI3K activation differs greatly among receptors,
depending on ligand binding kinetics and feedback circuitry that
can either amplify or dampen PI3K signaling (14). Additionally,
co-ligation of receptors, such as the T cell receptor (TCR) and the
CD28 co-receptor, can cooperate to potentiate and sustain PI3K
activation and PIP3generation.
PIP3ASSOCIATION WITH PLECKSTRIN HOMOLOGY
DOMAINS
PI3K activation induces PIP3accumulation, which comprises less
than 5% of PIP2levels and less than 1% of total membrane
lipids (15). Despite its low overall abundance, super-resolution
microscopy has revealed ~100nm membrane clusters of PIP3
that create high local PIP3concentrations (16). High affinity and
specificity binding between PIP3and pleckstrin homology (PH)
domains of PI3K effectors helps to recruit and activate these effec-
tors at the plasma membrane (Figure 2). Like protein–protein
interactions that are induced by phosphorylation, PIP3interac-
tions with PH domains allow rapid transduction of downstream
signals without new protein synthesis.
The PH domain is an evolutionarily conserved structural fold
found in proteins expressed in organisms ranging from yeast to
mammals (17). The core of the PH domain is a seven-strand
β-barrel that is encoded by approximately 120 amino acids and
is composed of two anti-parallel βsheets and a C-terminal
αhelix (Figure 3). The mammalian genome contains roughly
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Wang et al. Akt and Itk in lymphocytes
FIGURE 1 | Activation of class I PI3Ks by YxxM signaling subunits and
GPCRs. Membrane receptors that activate PI3K include CD19, CD28, and
NKG2D co-receptors, cytokine receptors (e.g., IL-2R), G-protein-coupled
receptors (chemokine receptors), and Fcγreceptor I and III. Class IA PI3Ks
are recruited to the plasma membrane through SH2 domain interactions with
phosphorylated YxxM motifs. Class IB PI3Ks are recruited and activated by
direct interaction with the Gβγ subunit following GPCR activation. Activated
PI3K phosphorylates the membrane lipid PI(4,5)P2to form PI(3,4,5)P3.
FIGURE 2 | PI(3,4,5)P3recruits PH domain-containing proteins to the
plasma membrane and regulates diverse cellular responses. PI3K
phosphorylates PI(4,5)P2to form PI(3,4,5)P3, which recruits PH
domain-containing signaling proteins to the plasma membrane. PH
domain-containing proteins are activated at the plasma membrane and
mediate important cellular responses such as cytoskeleton rearrangement,
cell growth, proliferation, and survival. PM, plasma membrane; GEF, guanine
nucleotide exchange factor.
300 PH domains found in proteins that perform diverse functions
including cellular activation, cytoskeletal reorganization, vesicu-
lar trafficking, and cell cycle control. Approximately, 15% of PH
domains, including Akt and Itk, bind to phosphoinositides with
high specificity and affinity (Kd: nanomolar – low micromolar
range). PH domains generally interact with phosphoinositides
through positively charged lysine and arginine residues within
the basic motif KXn(K/R)XR (18). However, not all PH domains
bind to PIP3. Several PH domains interact with phosphoinositides
that are selectively enriched in other membrane compartments,
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Wang et al. Akt and Itk in lymphocytes
FIGURE 3 | Crystal structure of Btk PH domain in complex with
Ins(1,3,4,5)P4(PDB ID: 2Z0P). The PH domain is comprised of a β-barrel
formed by seven β-strands (yellow, 1–7) capped by an α-helix (pink). The
hyper-variable loops of the β-barrel form the binding surface for lipid ligands
such as Ins(1,3,4,5)P4[top, shown by ball-and-stick model: red (oxygen),
orange (phosphorus), and gray (carbon)].
such as PI4P within the Golgi membrane (19) or PIP2at the
resting plasma membrane (17). Thus, conveying lipid speci-
ficity to PH domains constitutes a key mechanism for spatially
sequestering distinct effector proteins within cells. Regulating the
abundance of lipids either in resting or activated cells controls
basal and induced effector activity. Additionally, regulated pro-
duction of lipid ligands such as PIP3within specific membrane
nano-domains can induce polarized activation of downstream
effectors in a robust but transient manner. This is because PIP3
abundance is not only spatial restricted but also finely con-
trolled by receptor-induced PI3K-dependent PIP3generation and
by phosphatase and tensin homolog deleted on chromosome
10 (PTEN) and SH2 domain-containing inositol 50-phosphatase
(SHIP) phosphatase-dependent PIP3metabolism.
PROTEIN PHOSPHATASES INHIBIT PI3K ACTIVATION WHILE
INOSITOL PHOSPHATASES REDUCE PIP3LEVELS
PI3K signaling is negatively regulated at distinct steps in its sig-
naling cascade by both protein and lipid phosphatases. Protein
tyrosine phosphatases SHP-1 and SHP-2 inhibit PI3K activation
by preventing early receptor signaling and by directly down-
regulating PI3K activity, the latter of which is accomplished by
de-phosphorylation of phospho-tyrosine residues within signal
adapter proteins and PI3K regulatory subunits (71). Inhibitory
receptors that restrict lymphocyte activation through SHP-1 or
SHP-2 include inhibitory killer-cell immunoglobulin-like recep-
tors (KIR) on NK cells (72), CD22 on B cells (73), and CTLA-4
and PD-1 on T cells (74,75). Phosphorylated immunoreceptor
tyrosine-based inhibition motifs (ITIM) within the cytoplasmic
domains of KIRs, CD22, and CTLA-4 recruit SHP-1 and SHP-2
to prevent activating signals at the plasma membrane (72,74,75).
Persistent T cell activation signals can also be inhibited by SHP-1
and SHP-2 recruitment to the immunoreceptor tyrosine-based
switch motif (ITSM) in PD-1, an inhibitory receptor expressed
on chronically stimulated T cells (76,77). For a detailed dis-
cussion regarding the requirements of SHP-1 and SHP-2 in T
cell development, differentiation, and effector function, refer to
Ref. (78).
In T cells, CTLA-4 can also directly repress Akt signaling by
recruiting the Ser/Thr phosphatase PP2A (77), which dephospho-
rylates the T308 (79,80) and possibly S473 (79), residues required
for Akt activity. Thus, CTLA-4 utilizes a dual approach to antag-
onize CD28 and PI3K signaling: SHP-2-dependent inhibition of
TCR signaling by CD3εde-phosphorylation and PP2A-dependent
de-phosphorylation of Akt (74,77,81).
Lipid and inositol phosphatases also prevent PI3K effector acti-
vation. PTEN and SHIP both dephosphorylate membrane PIP3.
However, while PTEN converts PIP3back to its lipid precursor
PI(4,5)P2to prevent further activation of PI3K effectors, SHIP
converts PIP3into PI(3,4)P2, a lipid that retains the ability to
bind the Akt PH domain (82). In the latter case, subsequent
de-phosphorylation of PI(3,4)P2into PI(3)P by the inositol phos-
phatase, INPP4B is required to“turn off ” Akt membrane recruit-
ment (83). Inhibitory receptors including FcγIIB on B cells and
mast cells and Ly49A and Ly49C on NK cells contain ITIM motifs
that recruit SHIP through its SH2 domain (84,85). Membrane
receptors with cytosolic PDZ domains recruit PTEN to control
PIP3levels. Although the functional significance of PDZ domain-
containing receptors on lymphocyte activation requires additional
investigation, maintaining appropriate PTEN levels is crucial for
the control of immune cell homeostasis and function (86).
GENERAL AND CELL TYPE-SPECIFIC Akt FUNCTIONS
Akt belongs to the AGC family of Serine/Threonine kinases. The
three Akt isoforms are differentially expressed in various cell types
but are 77–83% sequence identical. Akt activity prevents apoptosis,
promotes protein expression, and regulates cellular metabolism
(2023). Akt mediates these general cellular functions through
direct phosphorylation of RxRxxS*/T* motifs (24) found in a
plethora of cellular targets including forkhead box transcription
factors, TSC2, GSK3, and BAD, which are discussed in detail else-
where (20). A somatic mutation in Akt that replaces glutamate with
lysine at residue 17 (hereafter referred to as E17K) leads to cellular
transformation and has been identified in human breast, colorec-
tal, and ovarian cancer (25,26). The E17K mutation is located
in the lipid binding pocket of Akt’s PH domain and dramatically
increases its affinity for membrane lipids, causing constitutiveAkt
signaling (27). Ectopic expression of E17K in hematopoietic stem
cells is sufficient to induce development of lymphoblastic T cell
lymphoma within 6–8 weeks following transfer into recipient mice
(28). Similarly, conditional deletion of the Akt targets Foxo1/3/4
in mice leads to development of the same type of lymphomas
15–25 weeks after induction of Foxo deletion (29).
In lymphocytes, Foxo proteins regulate the gene expression
of Rag recombinases, Ikaros, CCR7, IL-7R, TCF7, Eomes, and
Foxp3, which are critical for controlling lymphocyte development,
trafficking, and differentiation (3037). Akt phosphorylation of
Foxo1 and Foxo3 leads to their degradation and down-regulates
Foxo-dependent gene expression (31,38). Genetic ablation of
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Wang et al. Akt and Itk in lymphocytes
both Foxo1 and Foxo3 causes a multi-focal autoimmune dis-
ease due to defective Foxp3 expression and T regulatory (Treg)
cell specification and function (34). Similarly, retroviral expres-
sion of constitutively active myristoylated Akt (myrAkt) in
CD4CD8thymocytes impairs Treg development in vivo fol-
lowing intrathymic transfer. Importantly, the inhibitory effect of
myrAkt is on de novo but not established Foxp3 expression (39).
In contrast, broad expression of myrAkt as a transgene under the
control of the CD2 promoter leads to increased regulatory T cell
numbers in vivo and enhanced suppressive activity (40). Interest-
ingly, conventional CD4+T cells expressing transgenic myrAkt are
less responsive to TGFβsuppression and fail to differentiate into
the Th17 lineage in response to TGFβand IL-6 in vitro (40).
A proper balance of Akt activity is also required for appro-
priate CD8+T cell maturation, effector function, and memory
development (41). Uzel and colleagues recently published a study
on patients with somatic dominant active p110δ(a catalytic sub-
unit of PI3K) expression (42). T cell blasts from these patients
have increased phosphorylation of AKT at T308 and S473, a
decline in Foxo1, increased S6 activation, and glucose uptake.
This hyperactive Akt/mTORC1 axis causes CD8 T cells to pro-
liferate more vigorously, differentiate more readily into effector
cells, and undergo cellular senescence. Sustained Akt activity in
these patients also impairs development of CD8 memory T cells,
which require a metabolic “switch” from glycolysis to fatty acid
oxidation (41,43). Furthermore, defective CD8 responses result
in recurrent sinopulmonary infections and chronic viremia due to
Epstein-Barr virus (EBV) and/or cytomegalovirus (CMV) infec-
tion (42). Cantrell and coworkers published a surprising finding
demonstrating distinct roles for PDK1 and Akt in promoting cellu-
lar metabolism and effector responses of CD8 T cells, respectively
(44). T cells expressing a catalytically inactive p110δor treated
with an Akt inhibitor are defective for Akt T308 phosphorylation.
Akt-defective CD8 T cells proliferate normally in response to IL-
2 but are unable to express proper lymphoid homing receptors
and cytotoxic effector proteins (44). In contrast, conditional dele-
tion of PDK1, the upstream activator of Akt, leads to defective
glucose uptake and metabolism, resulting in reduced CD8 T cell
proliferation. This indicates that PDK1 promotes proliferation in
an Akt-independent manner (44). It remains to be determined
whether PDK1 and Akt have distinct roles in cell types in which
multiple functions have been attributed to Akt activity.
TEC FAMILY KINASES REGULATE IMMUNE CELL
DEVELOPMENT AND FUNCTION
The Tec family of non-receptor tyrosine kinases, including Tec, Btk,
Itk/Emt/Tsk, Rlk/Txk, and Bmx/Etk,are differentially expressed in
immune cells. Each Tec family member contains an N-terminal
PIP3-binding PH domain except Rlk, which contains a cysteine-
string motif that results in Rlk palmitoylation. In general, Tec
kinases activate PLCγto trigger Ca2+and diacylglycerol (DAG)
signaling. Mimicking Ca2+and DAG activation with the addition
of calcium ionophores and phorbol myristate acetate (PMA) is
sufficient to induce many aspects of lymphocyte activation, dif-
ferentiation, and effector responses in vitro. The requirement for
Tec kinases in immune functions is apparent from the profound
defects observed in human patients carrying mutations in Tec
kinases and in mouse models of single and combined Tec kinase
deficiencies.
In 1993, Btk was first identified in patients with X-linked
agammaglobulinemia (XLA), an inherited immunodeficiency dis-
ease characterized by profound hypogammaglobulinemia due to
severely decreased B cell numbers (45). XLA patients carry Btk
mutations that prevent the maturation of pro-B cells into pre-B
cells. Pre-B-cell receptor signaling at the pro-B to pre-B transi-
tion requires Btk activation by the Src kinase Lyn (4648). A
Btk mutation database generated from approximately 400 XLA
patients indicates that the majority of missense mutations in the
Btk PH domain are in the putative PIP3-binding pocket (49
51). The XLA missense mutants F25S, R28H, T33P, V64F, and
V113D dramatically reduce Btk binding to PIP3in vitro and dis-
rupt Btk activation in B cells (52,53). A similar mutation in mice,
R28C also abolishes Btk binding to PIP3and results in murine
X-linked immunodeficiency (Xid) disease (53). These findings
demonstrate the importance of PI3K-dependent PIP3generation
for the membrane recruitment and activation of Btk in promoting
B cell receptor signaling during maturation and humoral immune
responses.
While disruption of PIP3association causes hypo-B-cell
responses, enhanced PIP3association also leads to B cell dysfunc-
tion. The Btk E41K mutant significantly increases Btk PH domain
affinity for phosphoinositides and results in constitutive mem-
brane localization when expressed ectopically in COS-7 cells (52,
53). Btk E41K expression allows cytokine-independent growth of
the pro-B-cell line Y16 (54), demonstrating its gain-of-function
activity. However, mice expressing a Btk E41K transgene controlled
by the MHC class II locus are more severely B cell-deficient than
even Xid mice (55). Lack of IgMhigh cells in the bone marrow sug-
gest that constitutive Btk E41K activation leads to inappropriate
deletion of immature B cells by mimicking strong BCR signals that
promote apoptosis of auto-reactive B cells (55). Thus, appropriate
levels of Btk activation are critical for developmental progression
of B cells, productive B cell activation and differentiation, as well
as deletion of auto-reactive cells.
The first patients identified with Itk mutations were initially
diagnosed with Hodgkin’s lymphoma but subsequently character-
ized to have an underlying immunodeficiency disease that prevents
control of EBV-induced B cell proliferation (56). Itk-deficient
patients have decreased T cells (57), which are required to control
EBV infection and prevent viral reactivation from latently infected
B cells (58). Detailed characterization of Itk-deficient mice reveals
multiple requirements for Itk during T cell development,differen-
tiation, and function (59,60). Like Btk in B cells, Itk participates in
proximal antigen receptor signaling and is directly phosphorylated
by a Src family kinase, in this case Lck (61). Activated Itk phos-
phorylates PLCγ1, which induces IP3-dependent increased intra-
cellular Ca2+levels as well as DAG-mediated signaling (59,62,63).
Itk is required for efficient CD4+T cell differentiation toward the
Th2 and Th17 lineages (59). Itk-deficient mice cannot generate
protective Th2 responses in multiple infection models, including
Leishmania major,Nippostrongylus brasiliensis, and Schistosoma
mansoni (59,64). Defective Th2 differentiation is accompanied by
substantially reduced production of the Th2 cytokines IL-4, IL-
5, and IL-13 by Itk-deficient T cells (65,66). Itk is also required
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Wang et al. Akt and Itk in lymphocytes
for optimal production of the Th17 cytokine, IL-17A but not IL-
17F (67). The selective requirement for Itk in IL-17A production
is mechanistically linked to a requirement for the transcription
factor nuclear factor of activated T cells (NFAT) in IL-17A tran-
scription (64,67,68). Prolonged Itk activation maintains cytosolic
Ca2+levels to promote sustained calcineurin-dependent NFAT
nuclear translocation. Itk deficiency or suboptimal TCR signal-
ing restricts autoimmunity by biasing T cell differentiation from
the Th17 toward the regulatory T cell lineage (69). In addition,
autoimmune organ destruction can be limited by Itk-dependent
control of transendothelial migration and tissue infiltration of
effector T cells (70). Thus, mechanisms that regulate the mag-
nitude and kinetics of Itk activity in T cells are important for
induction of effector functions, specification of appropriate T cell
lineages, and control of T cell trafficking.
SOLUBLE ANALOGS OF PIP3DIFFERENTIALLY REGULATE PIP3
EFFECTORS
Some PIP3-binding PH domains can associate with soluble
PIP3analogs. These include the cytosolic inositol phos-
phates Ins(1,3,4,5)P4(IP4), Ins(1,2,3,4,5,6)P6(IP6), and 5-PP-
I(1,2,3,4,6)P5(IP7) that are generated inducibly or constitutively
by distinct inositol kinases (82). The effect of IP4, IP6, and
IP7binding is distinct for different PH domains and cell types
(Figure 4).
The inositol kinases IP3kinase (Itpk) isoforms A, B, and C,
and inositol polyphosphate multikinase (IPMK) can each gener-
ate IP4by phosphorylating Ins(1,4,5)P3(IP3) at the D-3 hydroxyl
group [reviewed in Ref. (87)]. However, mice deficient in the ubiq-
uitously expressed ItpkC or IPMK isoforms or in the neuronally
enriched ItpkA isoform have no detectable immune abnormalities.
In contrast, ItpkB expression is selectively enriched in hematopoi-
etic cells and catalytically activated by the Ca2+-sensing protein
calmodulin (CaM) following antigen receptor signaling. Analysis
of ItpkB-deficient mice revealed a non-redundant requirement for
ItpkB in lymphocyte development and activation (8892). ItpkB
deficiency results in severely reduced peripheral T cell numbers
due to an absolute block in positive selection of CD4+CD8+thy-
mocytes (88). Defective activation of the Ras/MAP kinase pathway
contributes to the T cell developmental defect (88,89,93). How-
ever, ItpkB-deficient CD4+CD8+thymocytes are also defective in
activation of Itk and its downstream effector PLC γ1 in response to
TCR engagement (93). Itk fails to localize to the plasma membrane
or assemble with the adapter protein LAT in the TCR signalo-
some of ItpkB-deficient thymocytes, indicating a requirement for
IP4in promoting Itk interactions (93). Interestingly, addition of
IP4increases binding of recombinant Itk PH domain to PIP3-
coated beads in vitro, suggesting that IP4may alter Itk PH domain
conformation to enhance PIP3accessibility (93).
Distinct from its effect on Itk, IP4suppresses Akt activity by
directly competing with PIP3for binding to the Akt PH domain
(94). ItpkB-deficient mice develop profound alterations in neu-
trophil and NK cell functions due to enhanced Akt activity during
their development and activation (94,95). Addition of membrane
permeable IP4, but not an isomer, to the myeloid cell line HL-60
disrupts membrane localization of an Akt PH domain fused to
GFP (94). In ItpkB-deficient neutrophils, Akt phosphorylation is
enhanced in response to the bacterial peptide Formyl–Methionyl–
Leucyl–Phenylalanine (fMLP). Enhanced Akt signaling in ItpkB-
deficient neutrophils contributes to augmented anti-microbialand
chemotaxis responses (94). The magnitude and kinetics of Akt
phosphorylation are also increased in ItpkB-deficient NK cells
FIGURE 4 | IP4and IP7negatively regulateAkt signaling. IP4and IP7are
cytosolic PIP3analogs that are able to associate with the Akt PH domain with
high affinity and can compete with membrane PIP3. IP4and IP7binding has
been proposed to dissociate Akt from the plasma membrane to prevent Akt
activation and substrate accessibility. IP4, Ins(1,3,4,5)P4; IP7,
5-PP-(1,2,3,4,6)IP5; PIP3, PI(3,4,5)P3.
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Wang et al. Akt and Itk in lymphocytes
(95). Elevated IFNγsecretion, granule exocytosis, and tumor cell
lysis by ItpkB-deficient NK cells can be suppressed by Akt inhi-
bition (95). Together, these studies indicate that IP4dampens
Akt activity in neutrophils and NK cells to restrict effector func-
tions. Whether this occurs to shut-off innate functions during the
resolution phase of an immune response or as a check to limit
inflammatory damage remains unclear.
Similar to IP4, IP7also competes with PIP3for binding to the
Akt PH domain and negatively regulates its activity (96). IP7is gen-
erated by pyro-phosphorylation of IP6at the 5-phosphate group
by IP6family kinases, IP6Ks (97,98). While the importance of
IP6K1 in lymphocyte function remains to be determined, analy-
sis of IP6K1-deficient neutrophils demonstrates similar functional
defects as ItpkB-deficient neutrophils. Both deficiencies result in
enhanced fMLP-induced chemotaxis, superoxide production, and
bacterial killing (94,99). Akt membrane localization and acti-
vation are significantly increased in IP6K1-deficient neutrophils
(99). Interestingly, IP7is readily detectable in resting HL-60 cells
but rapidly decreases upon fMLP stimulation (99). This suggests
that IP7may act to suppress initial Akt activation while IP4reg-
ulates subsequent Akt activity following its induced production.
Precise regulation of basal and induced IP4and IP7levels may
act together to control the magnitude and kinetics of Akt activa-
tion in these innate immune cells. Future studies are required to
determine the functional effects of IP4and IP7on Akt-dependent
regulation of lymphocyte differentiation and effector responses.
It also remains to be determined whether IP7acts on other PIP3
effectors in immune cells as it does in Dictyostelium discoideum
(100) or whether selective IP7binding allows regulation of a
particular subset of PIP3effectors.
Recently, biochemical and structural analyses of Btk identi-
fied a new activating function for the inositol phosphate, IP6
(101). As with PIP3-containing liposomes, addition of soluble
IP6induces Btk trans-phosphorylation and activation. However,
IP6promotes Btk activation by an unconventional mechanism
that is independent of the PIP3-binding pocket and membrane
recruitment. Analysis of the co-crystal structure of IP6with the
Btk PH domain reveals an additional peripheral IP6binding site
sandwiched between two PH modules, termed the Saraste dimer.
Molecular dynamics simulations suggest that IP6neutralizes elec-
trostatic forces in the monomer that oppose dimer formation.
Mutation of the IP6peripheral binding site disrupts transient
dimerization and significantly abrogates IP6-dependent Btk trans-
phosphorylation (101). IP6-induced Btk activation in solution
represents a new PI3K-independent mechanism for controlling
Btk activity. Considering that IP6levels are basally high in lym-
phocytes, it will be important in future studies to determine
whether IP6contributes to tonic or B cell receptor-induced Btk
function.
PROTEINS INTERACT WITH AND REGULATE THE ACTIVITY
OF PH DOMAIN-CONTAINING PROTEINS
Although the Akt and Itk PH domains specifically bind to PIP3
with (nanomolar) affinities, only ~40 mammalian PH domains
appear to be PIP3-regulated according to Teruel and colleagues,
who developed a prediction algorithm based on experimental
analyses of 130 mouse PH domains (102). The majority of PH
domains do not interact with lipids or bind lipids promiscuously
or with low affinity (Kd10 µM). Furthermore, a growing num-
ber of PH domains have been reported to participate in inter-
and/or intra-molecular protein interactions (discussed below).
These findings support a revised view of PH domains as diverse,
multifunctional domains that bind lipids, proteins, or both to
regulate the activity of their parent proteins.
T and B cells induce Ca2+and DAG-mediated signaling
through PLCγ1- and PLCγ2-mediated cleavage of PIP2(103,104).
T cell-specific ablation of PLCγ1 causes defects in thymocyte selec-
tion during T cell development, reduced T cell proliferation and
cytokine secretion, and the development of autoimmunity result-
ing from defective regulatory T cells (104). PLCγ2 plays important
roles in regulating B cells, neutrophils, mast cells, and dendritic
cells (105107). PLCγ1 and PLCγ2 both contain two PH domains.
The conventional, N-terminal PH domain associates with PIP3
(108); however, the C-terminal PH domain is interrupted by an
intervening amino acid sequence comprising two tandem SH2
domains and an SH3 domain (109,110). This split PH domain
is also critical for substrate binding (111). The C-terminal half of
the PLCγ1 split PH domain associates with a partial PH domain
in TRPC3 (112,113), a Ca2+channel that can mediate Ca2+entry
in T cells. The formation of this inter-molecular PH-like domain
allows PLCγ1 to bind to its substrate PIP2and is critical for TRPC3
membrane targeting and surface expression (113). Conversely, the
split PH domain of PLCγ2 interacts with the small GTPase Rac2,
which mediates PLCγ2 activation and localization to the plasma
membrane (114116).
Pleckstrin homology domains also participate in intra-
molecular interactions. In resting cells, the Akt PH domain asso-
ciates with the kinase domain (KD) to maintain a closed confor-
mation in which the activation loop is blocked (117,118). PIP3
binding to the Akt PH domain exposes the activation loop,allow-
ing T308 and S473 to be accessed and phosphorylated by PDK1
and mTORC2, respectively (119). Phosphorylation of T308 and
S473 fully activates Akt and keeps the activation loop open” for
substrate docking (117119). PH domain mutations that disrupt
PH–KD interaction (e.g., L52R and Q79K) result in constitutive
Akt activation (119).
The Dbl family RhoGEF Vav is also regulated by lipid and intra-
molecular interactions involving its PH domain (Figure 5). Vav
plays crucial roles during T cell and B cell development (120,121)
and T cell, B cell, neutrophil, and NK cell activation (9,107,120
123). Vav contains a Dbl homology (DH) domain that promotes
the activation of the small GTPase Rac in response to PI3K activa-
tion (124,125). In quiescent cells, Vav1 adopts an auto-inhibitory
conformation, which is stabilized by interactions between its PH,
acidic (Ac), and calponin homology (CH) domains (126,127). A
truncation mutation of the Vav N-terminal CH domain was shown
to have oncogenic potential (128), highlighting the importance of
these intra-molecular interactions in limiting Vav activity. Dur-
ing T cell activation, Lck phosphorylates tyrosine residues within
the Ac domain to release Vav1 from auto-inhibition (127). PIP3
binding to the PH domain significantly enhances Lck-dependent
Vav1 phosphorylation in vitro (129) and promotes GEF activity
(124,129,130) likely through the release of auto-inhibition (131).
Interestingly, PIP2binding to the Vav1 PH domain inhibits GEF
Frontiers in Immunology |T Cell Biology March 2015 | Volume 6 | Article 117 | 6
Wang et al. Akt and Itk in lymphocytes
FIGURE 5 | PH domain interactions stabilizeVav1 auto-inhibition in
basal state. In the basal state, Vav1 adopts an auto-inhibitory
conformation in which the substrate-docking site within the DH domain is
blocked by interactions with a helix region from the Ac domain. The
interactions between CH, PH, and Ac domains greatly strengthen the
auto-inhibitory conformation (left). During T cell activation, phosphorylation
of the Ac domain by Lck releases the substrate-docking site and allows
GTPase binding (right).
FIGURE 6 | CaM binds the Itk PH domain in a positive feedback loop that
potentiates Itk activity, intracellular Ca2+release, and IL-17A production.
Binding of Itk to PIP3promotes Itk activation and the subsequent
phosphorylation and activation of PLC γ1. PLCγ1 cleaves PIP2to produce DAG
and IP3, which binds IP3receptors on the ER.The IP3receptor is a
ligand-gated Ca2+channel, and its activation increases Ca2+levels in the
cytosol. Increased cytosolic Ca2+activates CaM, which has at least two
effects onT cell activation: (1) Ca2+/CaM binds to Itk’s PH domain, enhancing
its interaction with PIP3and Itk activity. (2) Ca2+/CaM binds to and activates
calcineurin, a phophatase that dephosphorylates NFAT, allowing NFAT
translocation to the nucleus where it drives the transcription of IL-17A. Thus,
CaM binding to Itk’s PH domain completes a positive feedback loop that
potentiates the downstream effects of Itk. PM, plasma membrane; ER,
endoplasmic reticulum; Itk, IL-2-inducible tyrosine/T cell kinase; PLCγ1,
phospholipase C gamma 1; CaM, calmodulin; NFAT, nuclear factor of activated
T cells; IP3R, IP3receptor.
activity (129). Thus, distinct lipids bind to the Vav1 PH domain to
promote conformational changes that either reinforce or relieve
its auto-inhibitory state.
Pleckstrin homology domains can also participate in inter-
molecular interactions with other proteins. The PH domain of
Dbs, a Cdc42/RhoGEF, associates with Cdc42 through the β3/β4
www.frontiersin.org March 2015 | Volume 6 | Article 117 | 7
Wang et al. Akt and Itk in lymphocytes
loop of its PH domain to improve substrate docking and catalysis
(132). Interestingly, we recently identified the β3/β4 loop of the
Itk PH domain as an important binding site for the ubiquitous
Ca2+-sensing protein CaM (133). The CaM C-terminal EF hands
bind to the β3/β4 loop of the Itk PH domain at basal intracellular
Ca2+levels while the CaM N-terminal EF hands engage the β5/β6
loop upon an increase in Ca2+levels. CaM and PIP3(but not IP4)
reciprocally enhance binding of one another to the Itk PH domain
in vitro, suggesting that CaM and PIP3cooperate to regulate Itk
signaling at the plasma membrane. Pharmacological inhibition
of Ca2+/CaM activity or mutation of the CaM-binding β3/β4
loop disrupts Itk-dependent activation of PLCγ1 and downstream
Ca2+responses (133), indicating that CaM participates in a posi-
tive feedback loop whereby binding of CaM to the Itk PH domain
enhances further Itk activation and downstream Ca2+responses.
Importantly, this positive feedback is required for optimal TCR-
induced, NFAT-dependent production of the pro-inflammatory
cytokine, IL-17A (133). Thus, CaM represents a novel protein-
binding partner for the Itk PH domain that serves an impor-
tant function in potentiating T cell pro-inflammatory responses
(Figure 6). It remains to be determined how CaM, PIP3, and IP4
coordinate to regulate the kinetics and magnitude of Itk activation
and whether they differentially participate in Itk-dependent T cell
activation, differentiation, and effector responses.
Calmodulin has also been reported by Dong and colleagues to
bind the PH domain of Akt family kinases (134). Using short pep-
tide fragments of Akt1 in a pulldown assay, this interaction was
further mapped to the first 42 residues of the Akt1 PH domain.
Although CaM did not directly alter Akt kinase activity, CaM was
reported to reduce the ability of PIP3to co-precipitate Akt (134),
suggesting that CaM competes with PIP3to dampen Akt activity.
However, this finding is inconsistent with other published data
demonstrating a requirement for CaM in optimal Akt phospho-
rylation at T308 and S473 (135,136). Thus, further investigation
is warranted to clarify the functional significance of CaM binding
to the AKT PH domain and to determine the precise role of this
interaction in lymphocytes.
CONCLUSION
The studies discussed herein highlight the essential yet complex
functions of PH domain-containing proteins in lymphocytes and
other immune cells. It is well established that a subset of PH
domains modulate the function of their parent proteins by binding
to membrane-bound lipids as well as soluble lipid analogs. Fur-
thermore, proteinsregulated in this manner, such as the PI3K effec-
tor kinases Akt and Itk,are indispensable for immune cell function.
Indeed, mutations that disrupt the lipid-binding capacity of PH
domains are known to result in human disease, a phenomenon
perhaps best demonstrated by the immunologic defects associ-
ated with mutations in Tec family kinases. Analogous and unique
pathological processes observed in animal models and in vitro
experiments reinforce the critical role of PH domain-containing
proteins in the immune system. However, evidence increasingly
shows that PH domains also interact with non-lipid substrates,
and these interactions can be cooperative, antagonistic, or com-
pletely independent of lipid-binding capacity. The breadth of these
interactions must be elucidated in order to fully understand role of
PH domain-containing proteins in immune cell function. Thus,
future work should investigate the capacity of PH domains to
interact with multiple substrates, including both lipids and pro-
teins, and should include careful evaluation of how binding of
each substrate affects the binding of others.
ACKNOWLEDGMENTS
This work was supported by NIH grant AI089805 to YH.
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Conflict of Interest Statement: The authors declare that the researchwas conducted
in the absence of any commercial or financial relationships that could be construed
as a potential conflict of interest.
Received: 12 January 2015; accepted: 02 March 2015; published online: 13 March 2015.
Citation: Wang X, Hills LB and Huang YH (2015) Lipid and protein co-
regulation of PI3K effectors Akt and Itk in lymphocytes. Front. Immunol. 6:117. doi:
10.3389/fimmu.2015.00117
This article was submitted to T Cell Biology, a section of the journal Frontiers in
Immunology.
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www.frontiersin.org March 2015 | Volume 6 | Article 117 | 11
... Pleckstrin Homology (PH) domains consist of around 120 amino acids and have a seven-strand β-barrel which forms 2 antiparallel β-sheets and a C-terminal α-helix ( Figure 1). The β1, β2, β3, and β4 loops form specific sites where the inositol ring of phosphoinositide binds (Wang et al., 2015). Approximately 10% of all PH domains possess high affinity and selectivity for phosphoinositides (Lemmon, 2007). ...
... New second messengers such as inositol trisphosphate (IP3) and diacylglycerol (DAG) are produced as a result of PLC activity on PtdIns(4,5)P2. Other kinases, such as PTENde, phosphorylates different sites on the inositol ring of PtdIns(3,4,5)P3 to produce PtdIns(4,5)P2 (Lemmon, 2007, Wang et al., 2015. While The PH domain consists of an α-helix (pink colour) and 7 β-strands (yellow colour), forming a β-barrel. ...
... PI binds to the PH domain through the loops of the β-barrel. (a) Btk-PH-Ins(1,3,4,5)P4 interaction; (b) PLC -PH PtdIns(4,5)P2 interaction (Bunney and Katan, 2011;Wang et al., 2015). the role of FA proteins PtdIns(4,5)P2 and PtdIns(3,4,5) P3 have previously been investigated, little attention has been given to quantify the local changes of PtdIns(4,5)P2 or PtdIns(3,4,5)P3 levels within and around FAs during their assembly and disassembly in MDA-MB-231 cells given the metastatic and high motility proprieties of the latter (Cailleau et al., 1974). ...
... Pleckstrin Homology (PH) domains consist of around 120 amino acids and have a seven-strand β-barrel which forms 2 antiparallel β-sheets and a C-terminal α-helix ( Figure 1). The β1, β2, β3, and β4 loops form specific sites where the inositol ring of phosphoinositide binds (Wang et al., 2015). Approximately 10% of all PH domains possess high affinity and selectivity for phosphoinositides (Lemmon, 2007). ...
... New second messengers such as inositol trisphosphate (IP3) and diacylglycerol (DAG) are produced as a result of PLC activity on PtdIns(4,5)P2. Other kinases, such as PTENde, phosphorylates different sites on the inositol ring of PtdIns(3,4,5)P3 to produce PtdIns(4,5)P2 (Lemmon, 2007, Wang et al., 2015. While The PH domain consists of an α-helix (pink colour) and 7 β-strands (yellow colour), forming a β-barrel. ...
... PI binds to the PH domain through the loops of the β-barrel. (a) Btk-PH-Ins(1,3,4,5)P4 interaction; (b) PLC -PH PtdIns(4,5)P2 interaction (Bunney and Katan, 2011;Wang et al., 2015). the role of FA proteins PtdIns(4,5)P2 and PtdIns(3,4,5) P3 have previously been investigated, little attention has been given to quantify the local changes of PtdIns(4,5)P2 or PtdIns(3,4,5)P3 levels within and around FAs during their assembly and disassembly in MDA-MB-231 cells given the metastatic and high motility proprieties of the latter (Cailleau et al., 1974). ...
... However, AKT activation also requires its phosphorylation at Ser473 by mTORC2 [45,46]. Activated AKT affects downstream targets, including GSK3, p21, FOXO, and mTOR, thus regulating multiple intracellular signaling pathways that affect cell growth, proliferation, differentiation, apoptosis, migration, secretion, angiogenesis, transcription, and protein synthesis [47,48]. IRSs are components of the downstream IIS pathway. ...
... However, AKT activation also requires its phosphorylation at Ser473 by mTORC2 [45,46]. Activated AKT affects downstream targets, including GSK3, p21, FOXO, and mTOR, thus regulating multiple intracellular signal-ing pathways that affect cell growth, proliferation, differentiation, apoptosis, migration, secretion, angiogenesis, transcription, and protein synthesis [47,48]. ...
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Circular RNAs (circRNAs) are a class of covalently circular noncoding RNAs that have been extensively studied in recent years. Aging is a process related to functional decline that is regulated by signal transduction. An increasing number of studies suggest that circRNAs can regulate aging and multiple age-related diseases through their involvement in age-related signaling pathways. CircRNAs perform several biological functions, such as acting as miRNA sponges, directly interacting with proteins, and regulating transcription and translation to proteins or peptides. Herein, we summarize research progress on the biological functions of circRNAs in seven main age-related signaling pathways, namely, the insulin-insulin-like, PI3K-AKT, mTOR, AMPK, FOXO, p53, and NF-κB signaling pathways. In these pathways, circRNAs mainly function as miRNA sponges. In this review, we suggest that circRNAs are widely involved in the regulation of the main age-related pathways and are potential biomarkers for aging and age-related diseases.
... Pleckstrin Homology (PH) domains consist of around 120 amino acids and have a seven-strand β-barrel which forms two anti-parallel β sheets and a C-terminal α helix. The β1, β2, β3 and β4 loops form specific sites to bind with the inositol ring of phosphoinositide (2). Approximately 10% of all PH domains have high affinity and specificity to bind with phosphoinositide (3). ...
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Full-text available
Background: Phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5) P3) and Phosphatidylinositol 4,5-trisphosphate (PtdIns(4,5) P2] form an insignificant amount of phospholipids but play important roles in controlling membrane-bound signalling. Little attention has been given to visualize and monitor changes or differences in the local generation of PtdIns(4,5) P2 and PtdIns(3,4,5) P3 in the cell membranes of MDA-MB-231 breast cancer cell lines. Methods: PLCδ1-PH-GFP and Btk-PH-GFP were used as biosensors to detected PtdIns(4,5) P2 and PtdIns(3,4,5)P3 respectively. These biosensors and antibodies were transfected, immuostained and then visualized by confocal microscopy on different cell surfaces. Results: Our results showed that PLCδ1-PH-GFP/mCherry was localized at the cell membrane, while Btk-PH-GFP/mCherry was sometimes localized at the cell membrane but there was also a large amount of fluorescence present in the cytosol and nucleus. Our results also showed that the cells that expressed low levels of Btk-PH-GFP the fluorescence was predominantly localised to the cell membrane. While the cells that expressed high levels of Btk-PH-GFP the fluorescence was localization in the cytosol and cell membrane. Our results demonstrated that both anti-PtdIns(4,5)P2 and anti-PtdIns(3,4,5)P3 antibodies were localized everywhere in cell. Conclusion: Our results suggest that PLCδ1-PH-GFP and Btk-PH-GFP/mCherry have more specificity, reliability, suitability and accuracy than antibodies in binding with and detecting PtdIns(4,5)P2 and PtdIns(3,4,5)P3 and in studying the molecular dynamics of phospholipids in live and fixed cells.
... Pleckstrin Homology (PH) domains consist of around 120 amino acids and have a seven-strand β-barrel which forms two anti-parallel β sheets and a C-terminal α helix. The β1, β2, β3 and β4 loops form specific sites to bind with the inositol ring of phosphoinositide (2). Approximately 10% of all PH domains have high affinity and specificity to bind with phosphoinositide (3). ...
Article
Full-text available
Background: Phosphatidylinositol 3,4,5-trisphosphate [PtdIns (3,4,5) P3) and Phosphatidylinositol 4,5-trisphosphate (PtdIns (4,5) P2] form an insignificant number of phospholipids but play important roles in controlling membrane-bound signalling. Little attention has been given to visualize and monitor changes or differences in the local generation of PtdIns (4,5) P2 and PtdIns (3,4,5) P3 in the cell membranes of MDA-MB-231 breast cancer cell lines. Methods: PLCδ1-PH-GFP and Btk-PH-GFP were used as biosensors to detected PtdIns (4,5) P2 and PtdIns(3,4,5)P3 respectively. These biosensors and antibodies were transfected, immuostained and then visualized by confocal microscopy on different cell surfaces. Results: Our results showed that PLCδ1-PH-GFP/mCherry was localized at the cell membrane, while Btk-PH-GFP/mCherry was sometimes localized at the cell membrane but there was also a large amount of fluorescence present in the cytosol and nucleus. Our results also showed that the cells that expressed low levels of Btk-PH-GFP the fluorescence was predominantly localised to the cell membrane. While the cells that expressed high levels of Btk-PH-GFP the fluorescence was localization in the cytosol and cell membrane. Our results demonstrated that both anti-PtdIns(4,5)P2 and anti-PtdIns(3,4,5)P3 antibodies were localized everywhere in cell. Conclusions: Our results suggest that PLCδ1-PH-GFP and Btk-PH-GFP/mCherry have more specificity, reliability, suitability and accuracy than antibodies in binding with and detecting PtdIns(4,5)P2 and PtdIns (3,4,5)P3 and in studying the molecular dynamics of phospholipids in live and fixed cells.
... Pleckstrin Homology (PH) domains consist of around 120 amino acids and have a seven-strand β-barrel which forms two anti-parallel β sheets and a C-terminal α helix. The β1, β2, β3 and β4 loops form specific sites to bind with the inositol ring of phosphoinositide (2). Approximately 10% of all PH domains have high affinity and specificity to bind with phosphoinositide (3). ...
Article
Full-text available
Background: Phosphatidylinositol 3,4,5-trisphosphate [PtdIns (3,4,5) P3) and Phosphatidylinositol 4,5-trisphosphate (PtdIns (4,5) P2] form an insignificant number of phospholipids but play important roles in controlling membrane-bound signalling. Little attention has been given to visualize and monitor changes or differences in the local generation of PtdIns (4,5) P2 and PtdIns (3,4,5) P3 in the cell membranes of MDA-MB-231 breast cancer cell lines. Methods: PLCδ1-PH-GFP and Btk-PH-GFP were used as biosensors to detected PtdIns (4,5) P2 and PtdIns(3,4,5)P3 respectively. These biosensors and antibodies were transfected, immuostained and then visualized by confocal microscopy on different cell surfaces. Results: Our results showed that PLCδ1-PH-GFP/mCherry was localized at the cell membrane, while Btk-PH-GFP/mCherry was sometimes localized at the cell membrane but there was also a large amount of fluorescence present in the cytosol and nucleus. Our results also showed that the cells that expressed low levels of Btk-PH-GFP the fluorescence was predominantly localised to the cell membrane. While the cells that expressed high levels of Btk-PH-GFP the fluorescence was localization in the cytosol and cell membrane. Our results demonstrated that both anti-PtdIns(4,5)P2 and anti-PtdIns(3,4,5)P3 antibodies were localized everywhere in cell. Conclusions: Our results suggest that PLCδ1-PH-GFP and Btk-PH-GFP/mCherry have more specificity, reliability, suitability and accuracy than antibodies in binding with and detecting PtdIns(4,5)P2 and PtdIns (3,4,5)P3 and in studying the molecular dynamics of phospholipids in live and fixed cells.
... PH domain is common between (PLCG1 and ITK proteins which show interactions in the GENE-MANIA analysis (S2 Fig). This conserved mammalian domain is responsible for the interaction between ITK and phosphoinositide 3-kinase (PI 3-kinase, PI3K) which in turn is the key player in lymphocyte differentiation and activation [60]. Computationally predicted functionally and structurally deleterious SNPs located in these regions could thus play an important role in this interaction (Fig 8). ...
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PLCG1 gene is responsible for many T-cell lymphoma subtypes, including peripheral T-cell lymphoma (PTCL), angioimmunoblastic T-cell lymphoma (AITL), cutaneous T-cell lymphoma (CTCL), adult T-cell leukemia/lymphoma along with other diseases. Missense mutations of this gene have already been found in patients of CTCL and AITL. The non-synonymous single nucleotide polymorphisms (nsSNPs) can alter the protein structure as well as its functions. In this study, probable deleterious and disease-related nsSNPs in PLCG1 were identified using SIFT, PROVEAN, PolyPhen-2, PhD-SNP, Pmut, and SNPS&GO tools. Further, their effect on protein stability was checked along with conservation and solvent accessibility analysis by I-mutant 2.0, MUpro, Consurf, and Netsurf 2.0 server. Some SNPs were finalized for structural analysis with PyMol and BIOVIA discovery studio visualizer. Out of the 16 nsSNPs which were found to be deleterious, ten nsSNPs had an effect on protein stability, and six mutations (L411P, R355C, G493D, R1158H, A401V and L455F) were predicted to be highly conserved. Among the six highly conserved mutations, four nsSNPs (R355C, A401V, L411P and L455F) were part of the catalytic domain. L411P, L455F and G493D made significant structural change in the protein structure. Two mutations-Y210C and R1158H had post-translational modification. In the 5’ and 3’ untranslated region, three SNPs, rs139043247, rs543804707, and rs62621919 showed possible miRNA target sites and DNA binding sites. This in silico analysis has provided a structured dataset of PLCG1 gene for further in vivo researches. With the limitation of computational study, it can still prove to be an asset for the identification and treatment of multiple diseases associated with the target gene.
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To the editor: Idiopathic CD4 lymphopenia represents a heterogeneous group of combined primary immunodeficiencies with markedly reduced CD4+ T-cell counts. Although several genetic etiologies including MHC class II deficiency[1][1] or mutations in RAG1 ,[2][2] MST1 ,[3][3] or LCK [4][4] have been
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