Figure 1: PIN1 deactivation/DAPK1 activation. When DAPK1
deactivates PIN1 and loses function of its ankyrin repeat domain (ARD),
it travels from the cytoskeleton to focal adhesion complexes. Through
this sub-localization of DAPK1, Src normally catalyzes its
phosphorylation for focal complex assembly to occur. In autism, due to
low expression of FAK and loss of PIN1 activation, Src function
predominates to assembly of matrix adhesion catalysis and impairs
cellular migration. Reduced cellular migration is an impairment
regularly seen in autism. Furthermore, increased cell adhesion complex
assembly contributes to normal cell adhesion and migration and cancer
cell death. On the other hand, anoikis initiated by DAPK1 localisation
to focal adhesion complexes is a causative factor for neurodegenerative
disease pathogenesis and a probable contributing factor for prion
disease and autism.
Preservation of homeostasis in adhesion complex formation has a
significant role in the prevention of neurodegeneration, as it maintains
the normal functions of neuronal synapses [165]. On a molecular
level, normal synaptic behavior is attributed to synaptic cell adhesion
molecule (CAM) pathways where the scaffolding SH3 ankyrin repeat domain
3 (SHANK3) proteins are the protagonists [166]. Dysregulation in
these CAM pathways involving neuroligins and neurexins, that regulate
cellular adhesions of synapses, are associated with the pathology of
autism spectrum disorders (ASDs) [167]. Multiple studies have shown
that many of the genes that are mutated in association with autism
involve proteins that play a critical role in cellular pathways
associated with the synapse. These genes encode scaffolding proteins,
adhesion molecules, and proteins involved in synaptic transmission and
plasticity. This observation has suggested that synaptic dysfunction may
be a core feature of autism [168]. DAPK1 is highly enriched in the
synaptic dendritic spines, where it plays an important regulatory role
in synaptic plasticity [169]. Furthermore, PIN1 localizes at
postsynaptic sites and, in particular, at the lipid rafts of dendrites
and postsynaptic density (PSD) areas which cohere to the postsynaptic
membrane and maintain synaptic plasticity [170,171].
The loss of PIN1 activity, as found in the brain cortical tissues of AD
patients, results in a decrease in SHANK protein levels and the
alteration of ubiquitin-related modifications of PSD proteins.
Structural abnormalities of the synapses of AD patients are a
consequence. Moreover, the loss of PIN1 activity due to oxidative stress
makes neurons more susceptible to the toxicity of amyloid-βfibrils, and this results in the inhibition of NMDA receptor stimulation
(and therefore reduced synaptic plasticity), as well as enhancement of
NMDA associated degeneration of synapses [171]. Constitutionally,
the loss of PIN1 activity and a concurrent operational activity of DAPK1
localised also at synaptic adhesion complexes of glutamate NMDA
receptors would provide a deregulated response of the otherwise
naturally occurring neuronal cell death by apoptosis [172]. In cases
where the extracellular matrix and the adhesion of cells fail to be
properly regulated, anoikis-induced cell death, including for neurons,
occurs [173]. These
mechanisms can feasibly explain many of the abnormal neural plasticity
features encountered in autistic individuals [168,173].
DAPK1 Regulatory Mechanisms and Protein Interactions: DAPK1 has
a profoundly complex regulatory mechanism involving phosphorylation and
dephosphorylation, and it interacts with many other important proteins
to alter their function in various ways. These other proteins include
not only PIN1, but also many biologically important proteins whose
disruption is linked to autism, including PP2A [174], ERK//MAPK
[175,176], and p53 [177], among others. One of the final
determinants of whether DAPK1 will be activated or not relies on the
phosphorylation of the serine 308 residue, which is located in the Ras
of complex (ROC) protein domain of the molecule, adjacent to the ankyrin
repeat domain. The ROC domain modulates its kinase activity. ROC is a
conserved domain of the ROCO multidomain family of proteins, making
DAPK1 a member of this family. ROC domains are implicated in the
development of neurodegenerative diseases, including Alzheimer’s disease
(AD) [178]. When the ROC domain of DAPK1 interacts with a GTPase,
the subsequent conversion of GTP to GDP phosphorylates Ser-308 and
inactivates the protein [86,177]. Nevertheless, this makes the
activation of DAPK1 subject to other ROCO protein kinase activations and
regulations [178]. In contrast, when the same Ser-308 becomes
dephosphorylated by the activity of protein phosphatase 2A (PP2A), an
important protein for cell signaling and growth mostly characterized as
a tumor suppressor, DAPK1 becomes activated [86,178,179] The
PP2A-DAPK1 interaction and activation via Ser-308 dephosphorylation in
the ROC subdomain is essential for many DAPK1 functions, including
apoptosis, regulation of autophagy and cellular proliferation and
development, and these are especially important cellular events for
halting cancer progression [180].
The ROC amino acid sequence of DAPK1, starting from residue 667, is
directly followed by the C terminal of ROC (COR) subdomain consisting of
the ROC-COR domain of DAPK1. This domain is then followed by the Death
Domain (DD), which is highly important for the molecule’s functions
[86]. The ROC-COR and DD domains of DAPK1 are highly significant for
regulation of PIN1 activity [181]. The 637–1423 sequence of DAPK1
efficiently binds to PIN1. This suggests that the DAPK1
cytoskeleton-binding domain, ranging from 637–847, which involves the
ROC subdomain, is highly likely involved with the binding of PIN1.
The peptide-to-peptide interactions between DAPK1 and PIN1 cause the
de-activation of PIN1’s isomerase activity by direct phosphorylation of
Ser71 in Pin1. Although, at first glance the phosphorylation of Ser71
seems to be a phenomenally minor molecular event, its biological and
medical consequences are of high impact for the survival of the cell
[86,180,181]. The destabilization and thus inhibition of cyclin D1
by DAPK1 is mediated by an additional functional insufficiency of PIN1.
The relevant experiments by TH Lee et al. are ominous [181]. When
DAPK1 is expressed in its wild form, PIN1 deactivation causes a cyclin
D1 destabilization and reduction in Cyclin D1 promoter activation.
Incapacitation of Cyclin D1 in the cells relates to severe deregulation
of cell cycle progression, amplification of cancerous molecular events
during cell cycling [182], and moreover, to the insufficiency of
neural cell proliferation and control that leads to autism [183].
Cyclin D1 levels are significantly reduced in various tissues of PIN1
NULL mice [184].
Furthermore, the ankyrin repeat, the ROC-COR and the DD domains of DAPK1
are subject to other molecular modifications that influence the
molecule’s pluripotent functions. When the tyrosine at positions 491/492
of the ankyrin repeat domain of DAPK1 is phosphorylated by the
proto-oncogene tyrosine-protein kinase Src (from sarcoma), this
deactivates DAPK1 from performing essential anti-cancer intra-molecular
interactions. The Src mediated deactivation of DAPK1 produces a crucial
loss of DAPK1-induced anti-cellular migration and proliferation
functions, and it is surely not coincidental that DAPK1 inactivation by
Src is found in situations of tumor metastasis and progression
[185].
However, as described above, DAPK1 over-activation and therefore PIN1
deactivation may be leading factors that contribute to autism brain
pathology by enhanced anoikis (apoptosis) events. FAK plays an essential
role in neuronal migration, neurite growth, pruning, and synapse
formation in the developing brain [186,187]. It has been proposed
that pathological detachment of progenitor cells during neurogenesis
induces anoikis as a defense mechanism to protect from teratogenic
insults [188]. This aligns with the observation that both glyphosate
and glyphosate-based herbicides produce teratogenic effects on Xenopus
laevis embryos and chicken embryos [189]. Plausibly, autistic
neurons, by having a reduced activity of FAK and therefore FAK/SRC
signaling, are defective in their migratory properties and therefore are
prone to enhanced cell death by anoikis during embryonic development.
Another major molecular event for DAPK1 is the phosphorylation of Ser735
located in the ROC-COR domain and the subsequent molecular interactions
with extracellular signal regulated kinase (ERK). The DAPK1 docking
sequence that serves as a substrate for ERK lies within the DAPK1 DD
[190]. The simultaneous induction of DAPK1 catalytic activity via
the activation of ERK promotes cellular death mechanisms including
neuronal apoptosis [191]. Moreover, interacting DAPK1 and ERK
mechanisms are involved in malignancy development and autism [108].
Finally, the DD of DAPK1 has a strong influence towards cancer
progression, as it is a direct modulator of p53 and a potent stimulator
of the TNF-α/Fas pathway resulting in apoptosis [159]. DAPK1
phosphorylates p53 at Ser23 by direct binding of DD at the p53 DNA
binding domain. Phosphorylation of p53 Ser23 by DAPK1 induces the
transcriptional activation of BAX, whereas in the cytoplasm it induces
mitochondrial associated necrosis and apoptosis, as indicated in studies
on neurons [191-194]. Moreover, an indirect regulation of p53 by
DAPK1 is through DAPK1’s direct binding to MDM2 and its phosphorylation
at Thr-419. This phosphorylation promotes MDM2 ubiquitination and
proteasomal degradation, liberates p53 from MDM2, and promotes p53
expression. The antitumorigenic associations of MDM2 downregulation and
indirect upregulation of p53 by DAPK1 are of considerable contemporary
interest in breast cancer research [195].