Zap70-Related SCID: Non-Redundant Dual Functions of the Zap70
Catalytic and Scaffolding Regions
Noah Isakov
The Shraga Segal Department of Microbiology and Immunology, Faculty of Health Sciences and the Cancer Research Center, Ben Gurion University of the Negev,
P.O.B. 653, Beer Sheva 84105, Israel
*Corresponding author:
Noah Isakov
The Shraga Segal Department of
Microbiology and Immunology
Ben-Gurion University
Beer-Sheva, Israel Tel: 972-8-6477267 Fax: 972-8-6477626 E-mail: noah@bgu.ac.il
Received March 21, 2012; Accepted March 24, 2012; Published March 28, 2012
Citation: Isakov N (2012) Zap70-Related SCID: Non-Redundant Dual Functions
of the Zap70 Catalytic and Scaffolding Regions. J Clinic Case Reports 2:e113.
doi:10.4172/2165-7920.1000e113
Primary Severe Combined Immunodeficiency (SCID) is a form
of heritable immunodeficiency, characterized by impaired adaptive
immune responses [1]. It includes a group of genetic disorders
originated by defects in one of several different genes that are critical
for T lymphocyte production and/or function and involve defects in B
lymphocytes as a primary or secondary cause [2].
The most common type of SCID is linked to the X chromosome
(X-SCID), and therefore affects only males [3,4]. The X-SCID males
possess mutations in the interleukin-2 (IL-2) receptor gamma chain
gene (IL-2R) encoding a protein that is shared by at least six different
receptors for interleukins, including IL-2 and IL-4. These receptors
direct the development and maturation of T and B lymphocyte subtypes
and help regulate the entire adaptive immune system.
Other forms of SCID follow an autosomal recessive inheritance
pattern that affects males and females equally. Among them, a relatively
rare genetic disorder is caused by mutations in a gene encoding the T
cell antigen receptor (TCR)/CD3-zeta chain (CD3ζ) associated protein
of 70 kDa (termed Zap70) [5-7], which is reflected by abnormal TCR
signaling.
Zap70-related SCID was first described in a patient of Mennonite
descent with CD8+ lymphocytopenia and normal numbers of CD4+ T
cells that did not proliferate in response to mitogenic stimulation [5-8].
A variety of distinct mutations in Zap70 were identified in different
patients, where the type of mutation determined the severity of the
immunodeficiency [9,10]. Affected children present in the first year of
life with recurrent bacterial, viral, and opportunistic infections, do not
usually survive past their second year without hematopoietic stem cell
transplantation.
Zap70 is a non-receptor protein tyrosine kinase (PTK) that plays
a critical role in TCR-linked signal transduction leading to T cell
differentiation and maturation and acquisition of effector function.
It was initially identified as a tyrosine phosphorylated protein with
a molecular mass of 70 kDa that associates with the CD3ζ chain of
activated T cells [11,12]. The transient binding of Zap70 to CD3ζ
involves the Zap70 tandem SH2 domains, which directly interact with
phosphorylated tyrosine residues within the immunoreceptor tyrosinebased
activation motifs (ITAMs) in the CD3ζ subunit [13].
The CD3ζ cytoplasmic tail possesses three copies of ITAMs,
each containing two tyrosine residues critical for its function. TCR
engagement by peptide-bound MHC molecules expressed on the
surface of antigen-presenting cells (APC) stimulates Lck, a member
of the Src family of PTKs, to phosphorylate the CD3ζ-ITAM-tyrosine
residues. The phosphorylated ITAMs serve as high affinity binding
sites for Zap70, thereby enabling its recruitment to T cell-APC contact
area, also termed the immunological synapse [14-17]. The tyrosine
phosphorylated CD3ζ-bound Zap70 undergoes phosphorylation by
Lck as well as autophosphorylation on specific tyrosine residues, which
alter its conformation and convert it into an active enzyme [18]. The
activated Zap70 then phosphorylates specific downstream molecules
[19-22], leading to calcium mobilization, activation of Ras GTPase and rearrangement of the actin cytoskeleton. These transient intracellular
signals permit the activation of selected transcription factors that
promote the proliferation and differentiation of T cells.
Analysis of the phosphorylation sites of ZAP-70 and their impact
on the function of the molecule demonstrated that phosphorylation
of Tyr319, Tyr492 and Tyr493 serves to regulate the Zap70 catalytic
activity [23-25]. However, in vivo activation of T cells results in Zap70
phosphorylation on additional tyrosine residues [26,27] which may
also function as putative docking sites for SH2-containing proteins.
Zap70 Tyr315 was reported to serve as a binding site for members of
the Crk family of adaptor proteins [27,28]. Furthermore, the CrkII-SH3
domain can interact with a variety of proteins that possess poly-proline
rich regions [29], thereby recruiting additional effector molecules to the
site of the engaged TCR. One CrkII-SH3 binding partner is the guanine
nucleotide exchange factor, C3G, which among other molecules,
regulates the GTPase Rap1 [30-32]. Activation of Rap1 increases the
inside-out signaling pathway that relays signals to the integrin LFA-1,
increasing its affinity to ICAM1, there by augmenting LFA-1/ICAM-1-
mediated T cell adhesion.
The above information and additional published studies indicated
that Zap70 possesses a catalytic region and a separate protein-protein
interaction (or scaffolding) region. Since distinct protein modules
within a single molecule might operate in a cooperative manner,
or independent of each other [33], it was of interest to decipher the
relationships between these two regions within the Zap70 molecule.
A recent study by Au-Yeung et al. [34] has solved this question
by showing that under certain conditions, the Zap70 protein exhibits
biological functions in regulatory T cells (Treg) that are independent of
the Zap70 catalytic activity.
To investigate the role of catalytically inactive Zap70 in normal
naïve and mature T cells, the authors have utilized a Zap70-deficient
mouse line in which they expressed a Zap70 mutant transgene that
retains catalytic activity yet can also be inhibited by a PP1 inhibitor
analog, termed 3-MB-PP1, a small-molecule kinase inhibitor [35].
The mutated analog-sensitive Zap70 protein, termed Zap70 (AS),
possesses a methionine-to alanine substitution at position 414 in the
ATP-binding domain, which allows it to accommodate the 3-MB-PP1
inhibitor. In addition, the 3-MB-PP1 molecule was found to specifically inhibit the Zap70 (AS) catalytic activity, and did not affect wild-type
Zap70 or other Src- or Tec-family PTKs [36].
When testing early activation events in Zap70(AS) and control
Zap70+/− CD4+ T cells stimulated by TCR/CD3 crosslinking, both
cell types responded by a robust increase in the intracellular free
Ca2+ concentration [Ca2+] and phosphorylation and activation of the
Erk MAPK. However, when TCR/CD3 crosslinking antibodies were
added to the cultured cells together with the 3-MB-PP1 inhibitor, a
dose-dependent inhibition of Ca2+ mobilization and Erk activation was
observed in Zap70(AS), but not in Zap70+/− CD4+ T cells. In addition,
Zap70(AS) and control Zap70+/− CD4+ T cells responded to TCR/CD3
plus CD28 crosslinking by strong proliferation, while Zap70(AS) but
not Zap70+/− CD4+ T cell proliferation was inhibited by 3-MB-PP1.
These results suggested that the catalytic activity of Zap70 is required
for both early and late activation of mature naïve CD4+ T cells.
Similar studies performed in antigen-primed T cells demonstrated
that interferon γ (IFN-) and IL-10 (IL-10) cytokine production by
antigen-challenged TH1 and TH2 CD4+ cells, respectively, require the
catalytic activity of Zap70. Furthermore, Zap70 catalytic activity
was required for the cytotoxic activity of primed alloreactive CD8+ T
cells, and for their ability to respond in tumor necrosis factor (TNF)
production.
A surprise came when the requirement for the Zap70 catalytic
activity was tested in the suppressive response of CD4+CD25+ Treg
cells [34]. In this assay, Au-Yeung et al. [34] have shown that TCR/
CD3-stimulated Zap70(AS) Treg cells that are cocultured with Zap70+/−
CD4+ CD25- conventional T cells (Tconv cells) have led to suppression
of the proliferative response of Zap70+/− Tconv cells. However, when
the suppressive activity of Zap70 (AS) Treg cells was tested, the cells
exhibited a similar inhibition of proliferation of Zap70+/− Tconv cells in
the absence but also in the presence of 3-MB-PP1, even when using
3-MB-PP1 concentrations that inhibited the anti-TCR/CD3-induced
proliferation of Zap70 (AS) Tconv cells. Although the TCR/CD3-
induced increase in [Ca2+] and Erk activation in Zap70(AS) Treg cells
were sensitive to suppression by 3-MB-PP1, the activation-induced
phosphorylation of Tyr319 and Tyr493 were not affected, apparently
because they serve as substrates for phosphorylation by Lck, which is
insensitive to 3-MB-PP1.
The results by Au-Yeung et al. [34] support previous findings
showing that tyrosine phosphorylated Zap70 can act as a scaffolding
protein [27, 28, 37, 38], and suggest that this activity is independent of
the Zap70 catalytic activity.
Au-Yeung et al. [34] have further demonstrated that CrkII and
C3G coimmunoprecipitate with activated Zap70 from Zap70 (AS)
Treg cells, even in the presence of 3-MB-PP1, and that activation of the
downstream effector molecules, C3G and Rap1, was also independent
of the catalytic activity of Zap70 [38]. Furthermore, TCR/CD3-induced
adhesion of Zap70 (AS) CD4+ T cells to ICAM-1-coated surface was not
sensitive to inhibition by 3-MB-PP1, suggesting that the TCR/CD3-
induced inside-out signaling that regulate T cell adhesion requires
the scaffolding function of Zap70, but is independent of the Zap70
catalytic activity. The results indicate that Zap70 is a candidate drug
target in autoimmune diseases and allograft rejection, and suggest that
different Zap70-specific drugs may vary in their ability to modulate
responses of distinct T cell subtypes.
Acknowledgement
Work in our laboratory is funded in part by the USA-Israel Binational Science
Foundation and the Israel Science Foundation administered by the Israel Academy
of Science and Humanities. N.I. holds the Joseph H. Krupp Chair in Cancer
Immunobiology.
OMICS Publishing Group is the member of / publishing partner of/source content provider to
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