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Structure of FAK family kinases
FAK and Pyk2 are composed of three conserved domains (Figure 4): an amino-terminal four-point-one, ezrin, radixin, moesin (FERM) domain, a central kinase domain and carboxy-terminal focal adhesion targeting (FAT) domain (Girault et al., 1999b; Lipinski and Loftus, 2010; Hall et al., 2011; Walkiewicz et al., 2015). These three domains are connected by two linker regions which contain proline-rich (PR) motifs. Unlike many nRTKs, neither FAK nor Pyk2 encompass Src-homology domains 2 and 3 (SH2 and SH3). FERM and FAT domains both contribute to the regulation of the enzymatic activity of FAK and Pyk2 and allow their interaction with many proteins playing a key role in signal transduction.
Figure 4. Comparison of human FAK and Pyk2 structures. FERM, kinase, FAT and PR domains, as well the main phosphorylated tyrosines are indicated. Percentages of identity (aminoacids) of each domain are specified.
FERM domain
FERM domains are roughly 300 amino-acids domains commonly found in proteins that bind cytoplasmic regions of transmembrane proteins and often act as linker between the cytoskeleton and plasma membrane (Chishti et al., 1998; Girault et al., 1999b; Riggs et al., 2011). Besides, FERM domains can also mediate intramolecular interactions. For instance, the functional activity of the prototypical FERM domain proteins ezrin, radixin, and moesin is regulated by FERM domain mediated intramolecular associations (Pearson et al., 2000; Edwards and Keep, 2001). The FERM domain has three lobes, namely: F1, F2 and F3, together forming a cloverleaf-shaped structure that mediates both protein-membrane targeting as well as protein-protein interactions (Figure 5). It is worth noting FAK FERM domain shares only 12²15% identity with the sequences of other FERM domains but they adopt a quite similar tertiary structure as shown by hydrophobic cluster analysis (Girault et al., 1999b).
FERM proteins are targeted to the membrane due to the interaction between basic residues in a cleft between subdomains F1 and F3 and PIP2 (Hirao et al., 1996; Hamada et al., 2000). This interaction further induces conformational changes of FERM proteins that would stimulate their interaction with the cytoplasmic tails of transmembrane proteins (Hamada et al., 2003).
The FERM domain also appears be a domain of interaction with various cytosolic and nuclear proteins. Among them, the three membrane-associated phosphatidylinositol transfer proteins (PITPNMs), a family of protein associated with metastasized cancers, interact with the FERM domain of Pyk2 but not FAK (Lev et al., 1999). In contrast, the transcription factor p53 was shown to interact with the FERM domain of both FAK (Golubovskaya and Cance, 2011) and Pyk2 (Lim et al., 2010). The mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) was shown to interact with the FERM domain of Pyk2 but not of FAK (Loftus et al., 2013).
Another function of the FERM domain is a potential autoregulatory role due to interactions with the kinase domain. This autoinhibitory interaction has been described in FAK (Lietha et al., 2007), and also, more recently, in Pyk2 (Loving and Underbakke, 2019). Moreover, in both FAK and Pyk2, the FERM domain is supposed to be mandatory for the activation-induced homodimerization (Kohno et al., 2008; Riggs et al., 2011; Brami-Cherrier et al., 2014).
Finally, a nuclear localization sequence (NLS) and a nuclear export signal (NES) were found in the F2 and F1 subdomain respectively of both FAK and Pyk2 showing a particular standing of this region for FAK family kinases nucleocytoplasmic shuttling (Lim et al., 2008a; Ossovskaya et al., 2008).
Cellular localization of FAK family kinases
In adhering non-neuronal cells, FAK is located in focal adhesions which are sub-cellular complexes forming mechanical links between intracellular actin bundles and the extracellular matrix (Schaller et al., 1992). Pyk2 is essentially located in the perinuclear region (Sieg et al., 1998; Avraham et al., 2000) but can be found, to a lesser extent than FAK, in focal adhesions (Du et al., 2001). In neurons, FAK is enriched is cellular body, growth cones and dendrites (Renaudin et al., 1999). Moreover, FAK can be enriched along perinuclear microtubules associated to centrosomes (Xie et al., 2003). In neurons, Pyk2 is enriched in cell body and dendritic shafts (Menegon et al., 1999; Corvol et al., 2005).
However, FAK and Pyk2 can also shuttle to the nucleus. Indeed, FAK was observed in the nucleus of cardiomyocytes under some pathological conditions (Lobo and Zachary, 2000; Yi et al., 2003). Pyk2 was also observed in the nucleus of various cell types including keratinocytes (Schindler et al., 2007), chondrocytes (Arcucci et al., 2006), and in depolarized neurons or PC12 cells (Faure et al., 2007). The dynamic of nucleocytoplasmic shuttling of FAK and Pyk2 results from the presence of multiple regulatory sequences detailed above. For instance, in the case of Pyk2, it was shown that the NES located on linker 2 was regulated by phosphorylation at residue Ser-778, a substrate of 3·,5·-cyclic adenosine monophosphate (cAMP)-dependent protein kinase and calcineurin, a Ca2+/calmodulin-activated protein phosphatase. The detailed mechanism of this regulation will be discussed in III.1.2.
Isoforms of FAK family kinases
Both FAK and Pyk2 present various isoforms resulting from alternative splicing of their messenger ribonucleic acid (mRNA) or from alternative transcription initiation sites (Figure 8).
FAK isoforms
To date, FAK possesses four additional peptides (called boxes) which can be included or not to the sequence and are named according to the number of aminoacids contained in the box: box 28, box 6, box 7 and box 3 (or PWR, based on the single letter amino acid code for three residues of this box, Pro, Trp, and Arg; FAK isoform containing this exon is termed FAK+).
These inserts provide biological differences between isoform. Besides, every isoform present a specific expression pattern. For instance the isoform containing the boxes 3, 6 and 7 (called FAK+6,7) is predominant in neurons (Burgaya et al., 1997).
Moreover, FAK gene possesses a second promotor located in the intron following the last exon coding for the catalytic domain. Expression of the C-ter domain from this promotor produces the so-called FAK-related non-kinase (FNRK). This variant is an in vitro inhibitor of FAK as it competes with FAK for the localization to focal adhesions by its intact FAT domain (Schaller, 2010). FRNK was shown to have physiological functions. Increased expression of FRNK prevented metastatic adhesion of hepatocellular carcinoma cells (von Sengbusch et al., 2005). Moreover, FRNK negatively regulates IL-4-mediated inflammation by attenuating eosinophil recruitment in a way that seems independent of FAK pathway (Sharma et al., 2015).
Pyk2 isoforms
Pyk2 has an isoform lacking 42 amino acids in the linker 2 (Dikic et al., 1998; Xiong et al., 1998; Keogh et al., 2002; Kacena et al., 2012). This isoform, called Pyk2-H (hematopoietic) or Pyk2-S (short), is abundantly expressed in hematopoietic cells where it is implied in cell response to some chemokines.
As for FAK, another isoform of Pyk2 results from the expression of the C-terminal domain only and is termed Pyk2-related non-kinase (PRNK) (Xiong et al., 1998). This variant also inhibits Pyk2 function and was tested for this reason in many experiments. Thus, exogenous expression of PRNK could prevent myocardial fibrosis (You et al., 2015), impair CD11b/CD18-mediated phagocytosis in macrophages (Paone et al., 2016), and could reduce squamous carcinoma cells viability, migration, invasiveness and adhesion ability (Yue et al., 2015). However, PRNK may act as an inhibitor of Pyk2 only in some cell types as it does not interact with the Pyk2 partner p130Cas or Graf (Xiong et al., 1998).
Finally, Arcucci et al discovered a nuclear fragment of Pyk2 of 68 kDa in chick embryo epiphyseal chondrocytes which is not recognized by an antibody raised against the N-terminal region of Pyk2 suggesting that this fragment lacks the N-terminal region (Arcucci et al., 2006). However, neither the mechanism of formation of this fragment nor its biological function is known.
Cellular functions of FAK
Among the adhesion sites mediated by integrins, the focal adhesions are long flat structures often located in the periphery of cells (Sastry and Burridge, 2000). They are mainly composed of integrin, paxillin, vinculin, talin and FAK.
Upon engagement of integrins by extracellular matrix, FAK is recruited to the focal adhesions and rapidly phosphorylated on tyrosine (Hanks et al., 1992; Kirchner et al., 2003; Zaidel-Bar et al., 2003). Activated FAK can interact with Src which phosphorylate many partners such as ơ-actinin which then recruits vinculin and regroups the actin bundles to the focal adhesions (Mitra et al., 2005) (Figure 9). FAK-/- fibroblasts have reduced and immature focal adhesions with a default of binding to the actin bundles showing that FAK is essential to the function of these structures (Iliý et al., 1995; Katoh, 2017). Particularly, loss of FAK results in the persistence of old focal adhesions and in the inability to form new ones showing the importance of FAK in focal adhesion turnover (Ren et al., 2000).
Physiological functions of FAK
FAK knockout mice is embryonic lethal around E8.5 showing a major role in development (Furuta et al., 1995). This phenotype principally results of a migration defect as detailed in the previous part (Iliý et al., 1995). Interestingly the function of FAK during early development involves more than its autophosphorylation mechanism. For example mice homozygous for a deletion of the exon that codes for Tyr-397 have a normal development up to E12.5 and die later (Corsi et al., 2009). FAK is also important in the nervous system in vivo as shown by several studies. It is highly expressed during brain development whereas its levels decrease in the adult (Burgaya et al., 1995). Conditional deletion of FAK in the cortex demonstrated its major role in basal lamina formation (Beggs et al., 2003) and in dendritic shaft formation (Gorski et al., 2002). Conforming to its role in focal adhesion turnover, FAK was shown to be a negative regulator of axonal branching and synapse formation (Rico et al., 2004). FAK is also critical for neuronal migration and axon guidance during development (Xie et al., 2003; Li et al., 2004b; Bechara et al., 2008; Chacón et al., 2012; Kerstein et al., 2017) as well as for the control of cortical dendrite arborization (Garrett et al., 2012). Additionally, conditional deletion of FAK in Schwann cells proved the requirement of FAK for axonal myelination (Grove and Brophy, 2014).
According to these various physiological functions, FAK is involved in many diseases such as cancer (Naser et al., 2018), ischemia (Bikis et al., 2015; Gao et al., 2018), beta peptide accumulation (Zhang et al., 1996; Williamson et al., 2002) and cardiac hypertrophy (Mohanty and Bhatnagar, 2018) and is thus studied as a potential target for many molecular therapies.
Table of contents :
Acknowledgments
Contents
List of abbreviations
Context and objectives
INTRODUCTION
Tyrosine kinase signaling and the FAK family
1. The origin of TKs and FAK family
2. FAK family
Identification of FAK family kinases
Structure of FAK family kinases
2.2.1. FERM domain
2.2.2. Linker 1
2.2.3. Kinase domain
2.2.4. Linker 2
2.2.5. FAT domain
Expression of FAK and Pyk2
Cellular localization of FAK family kinases
Isoforms of FAK family kinases
2.5.1. FAK isoforms
2.5.2. Pyk2 isoforms
Biological functions of FAK
2.6.1. Cellular functions of FAK
2.6.2. Physiological functions of FAK
Pyk2: a nRTK of FAK family
1. Regulation of Pyk2 activity in non-neuronal cells
Activation and phosphorylation of Pyk2
1.1.1. Canonical activation of Pyk2
1.1.2. Regulation of Pyk2 activation by Ca2+-activated kinases
1.1.2.1. PKC
1.1.2.2. CaMKII
Dephosphorylation of Pyk2
1.2.1. Tyrosine phosphatases
1.2.1.1. SHP-1
1.2.1.2. SHP-2
1.2.1.3. PTP-PEST
1.2.1.4. STEP
1.2.2. Ser/Thr phosphatases
SUMOylation of Pyk2
S-nitrosylation of Pyk2
2. Pyk2 functions in non-neuronal cells
Pyk2 cellular functions
2.1.1. Cell adhesion
2.1.2. Cell migration
2.1.3. Cell division
2.1.4. Cell survival
2.1.5. Cell differentiation
Physiological role of Pyk2
2.2.1. Bone physiology
2.2.2. Vascular system integrity
2.2.3. Immune system function
2.2.4. Kidney function
2.2.5. Sperm capacitation
2.2.6. Generation of Pyk2 knockout mice
3. Pathological role of Pyk2
Pyk2 and inflammatory diseases
Pyk2 and cancers
Pharmacological inhibitors of Pyk2
Roles of Pyk2 in the CNS
1. Specific regulation of Pyk2 in the CNS
Activation of Pyk2 in neurons
Regulation of Pyk2 localization
2. Pyk2 biological functions in the CNS
Ionic channels regulation
2.1.1. Kv1.2
2.1.2. BK channels
2.1.3. NMDA receptor (NMDAR)
Development
Synaptic plasticity
Neuronal survival
Pyk2 in glial cells
3. Pyk2 in CNS diseases
Alzheimer·s disease
Parkinson·s disease
Huntington·s disease
Neuroinflammation
Glioma and neuroblastoma
Cerebral ischemia
Psychiatric disorders
RESULTS
Pyk2 modulates hippocampal excitatory synapses and contributes to cognitive deficits in a Huntington’s disease model
1. Context and objectives
2. Contribution to the work
3. Article
4. Summary of the findings and conclusions
Pyk2 in the amygdala modulates chronic stress sequelae via PSD-95-related microstructural changes
1. Context and objectives
2. Contribution to the work
3. Article
4. Summary of the findings and conclusions
PTK2B/Pyk2 overexpression improves a mouse model of Alzheimer’s disease
1. Context and objectives
2. Contribution to the work
3. Article
4. Summary of the findings and conclusions
Conditional BDNF Delivery from Astrocytes Rescues Memory Deficits, Spine
Density, and Synaptic Properties in the 5xFAD Mouse Model of Alzheimer Disease.
1. Context and objectives
2. Contribution to the work
3. Article
4. Summary of the findings and conclusions
Pyk2 in nucleus accumbens D1 receptor-expressing neurons is selectively involved in the acute locomotor response to cocaine
1. Context and objectives
2. Contribution to the work
3. Article
4. Summary of the findings and conclusions
Supplementary data: spine density and morphology in the NAc of Pyk2-/- mice
1. Materials and methods
2. Results
DISCUSSION
Role of Pyk2 in memory
Kinase-dependent and independent functions of Pyk2
Antagonistic effect of Pyk2 on spine density and morphology
BDNF and Pyk2 merging functions
Pyk2 and AD: risk or rescue factor?
Contrasted function of Pyk2 in the striatum
BIBLIOGRAPHY
