Gene name - p21-activated kinase
Synonyms - PAK-kinase
Cytological map position - 83E
Function - signaling
Symbol - Pak
FlyBase ID: FBgn0267698
Genetic map position - 3-
Classification - PAK family kinase
Cellular location - cytoplasmic
|Recent literature||Wang, M., Chen, P. Y., Wang, C. H., Lai, T. T., Tsai, P. I., Cheng, Y. J., Kao, H. H. and Chien, C. T. (2016). Dbo/Henji modulates synaptic dPAK to gate glutamate receptor abundance and postsynaptic response. PLoS Genet 12: e1006362. PubMed ID: 27736876
In response to environmental and physiological changes, the synapse manifests plasticity while simultaneously maintains homeostasis. This study analyzed mutant synapses of henji, also known as dbo, at the Drosophila neuromuscular junction (NMJ). In henji mutants, NMJ growth is defective with appearance of satellite boutons. Transmission electron microscopy analysis indicates that the synaptic membrane region is expanded. The postsynaptic density (PSD) houses glutamate receptors GluRIIA and GluRIIB, which have distinct transmission properties. In henji mutants, GluRIIA abundance is upregulated but that of GluRIIB is not. Electrophysiological results also support a GluR compositional shift towards a higher IIA/IIB ratio at henji NMJs. Strikingly, dPAK, a positive regulator for GluRIIA synaptic localization, accumulates at the henji PSD. Reducing the dpak gene dosage suppresses satellite boutons and GluRIIA accumulation at henji NMJs. In addition, dPAK associated with Henji through the Kelch repeats which is the domain essential for Henji localization and function at postsynapses. It is proposed that Henji acts at postsynapses to restrict both presynaptic bouton growth and postsynaptic GluRIIA abundance by modulating dPAK.
|Aguilar-Aragon, M., Elbediwy, A., Foglizzo, V., Fletcher, G. C., Li, V. S. W. and Thompson, B. J. (2018). Pak1 kinase maintains apical membrane identity in epithelia. Cell Rep 22(7): 1639-1646. PubMed ID: 29444419
Epithelial cells are polarized along their apical-basal axis by the action of the small GTPase Cdc42, which is known to activate the aPKC kinase at the apical domain. However, loss of aPKC kinase activity was reported to have only mild effects on epithelial cell polarity. This study shows that Cdc42 also activates a second kinase, Pak1, to specify apical domain identity in Drosophila and mammalian epithelia. aPKC and Pak1 phosphorylate an overlapping set of polarity substrates in kinase assays. Inactivating both aPKC kinase activity and the Pak1 kinase leads to a complete loss of epithelial polarity and morphology, with cells losing markers of apical polarization such as Crumbs, Par3/Bazooka, or ZO-1. This function of Pak1 downstream of Cdc42 is distinct from its role in regulating integrins or E-cadherin. These results define a conserved dual-kinase mechanism for the control of apical membrane identity in epithelia.
|Biehler, C., Rothenberg, K. E., Jette, A., Gaude, H. M., Fernandez-Gonzalez, R. and Laprise, P. (2021). Pak1 and PP2A antagonize aPKC function to support cortical tension induced by the Crumbs-Yurt complex. Elife 10. PubMed ID: 34212861
The Drosophila polarity protein Crumbs is essential for the establishment and growth of the apical domain in epithelial cells. The protein Yurt limits the ability of Crumbs to promote apical membrane growth, thereby defining proper apical/lateral membrane ratio that is crucial for forming and maintaining complex epithelial structures such as tubes or acini. This study shows that Yurt also increases Myosin-dependent cortical tension downstream of Crumbs. Yurt overexpression thus induces apical constriction in epithelial cells. The kinase aPKC phosphorylates Yurt, thereby dislodging the latter from the apical domain and releasing apical tension. In contrast, the kinase Pak1 promotes Yurt dephosphorylation through activation of the phosphatase PP2A. The Pak1-PP2A module thus opposes aPKC function and supports Yurt-induced apical constriction. Hence, the complex interplay between Yurt, aPKC, Pak1, and PP2A contributes to the functional plasticity of Crumbs. Overall, these data increase understanding of how proteins sustaining epithelial cell polarization and Myosin-dependent cell contractility interact with one another to control epithelial tissue architecture.
|Gao, J., Zhao, B. R., Zhang, H., You, Y. L., Li, F. and Wang, X. W. (2021). Interferon functional analog activates antiviral Jak/Stat signaling through integrin in an arthropod. Cell Rep 36(13): 109761. PubMed ID: 34592151
Drosophila Vago is a small antiviral peptide. Its ortholog in Culex mosquito was found to be an interferon-like cytokine that limits virus replication through activating Jak/Stat signaling. However, this activation is independent of Domeless, the sole homolog of vertebrate type I cytokine receptor. How Vago activates the Jak/Stat pathway remains unknown. Herein, this process is dependent on integrin in kuruma shrimp (Marsupenaeus japonicus). Shrimp Vago-like (MjVago-L) plays an antiviral role by activating the Jak/Stat pathway and inducing Stat-regulated Ficolin. Blocking integrin abrogates the role of MjVago-L. The interaction between MjVago-L and integrin β3 is confirmed. An Asp residue in MjVago-L is found critical for the interaction and MjVago-L's antiviral role. Moreover, Fak, a key adaptor of integrin signaling, mediates MjVago-L-induced Jak/Stat activation. Therefore, this study reveals that integrin, as the receptor of MjVago-L, mediates Jak/Stat activation. The establishment of the MjVago-L/integrin/Fak/Jak/Stat/Ficolin axis provides insights into antiviral cytokine signaling in invertebrates. (Gao, 2021)
Paks (p21-activated kinases) are evolutionarily conserved regulators of the actin cytoskeleton. Drosophila PAK kinase, initially isolated on the basis of its homology to other PAK proteins, interacts with the small GTP-binding proteins Rac and Cdc42 and functions in signal transduction during dorsal closure (Harden, 1996). The yeast Pak Ste20 regulates polarized cell growth in response to mating pheromones, and mammalian Paks reorganize the actin cytoskeleton when overexpressed in tissue culture cells. Pak consists of an N-terminal regulatory region that inhibits the activity of the C-terminal kinase domain. The regulatory region contains binding sites for at least three signaling proteins: an N-terminal proline-rich sequence (PXXP) binds to Nck; a CRIB (Cdc42/Rac interactive binding) motif binds to GTP-bound forms of Cdc42 and Rac, and a proline-rich motif is constitutively bound to Pix, a guanine nucleotide exchange factor specific to Rac and Cdc42. In the inactive state, an autoinhibitory sequence adjacent to the CRIB motif inhibits Pak kinase activity (Hing, 1999 and references).
These biochemical studies have provided a basis for a model of Pak function in growth cones. In this model, recruitment of the Pak/Pix complex to the membrane by mammalian Nck (Drosophila homolog Dreadlocks) in response to guidance receptor activation promotes GTP binding and activation of the p21 GTP binding proteins Cdc42 and Rac by Pix. Cdc42/Rac can, in turn, bind the CRIB motif of Pak and induce a conformational change in Pak. This displaces the autoinhibitory peptide, thereby activating Pak kinase, which also explains the name of this group: p21 activated kinases, or PAKs. Hence, recruitment of the Pak/Pix complex to the membrane may lead to the activation of both Rho family GTPases and Pak (Hing, 1999 and references).
Dreadlocks (Dock), an SH3/SH2 adaptor protein, links guidance signals to changes in the actin-based cytoskeleton in photoreceptor (R cell) growth cones (Garrity, 1996). The compound eye of the fly contains some 800 simple eyes called ommatidia. Each ommatidium contains eight R cells (R1-R8) that project in a retinotopic fashion to two different layers in the brain. R1-R6 terminate in the lamina, whereas R7 and R8 terminate in the medulla. While dock mutant R cells extend axons into the optic lobe normally, they form abnormal patterns of connections in both the lamina and medulla with defects in topographic map formation and ganglion target specificity (i.e., lamina versus medulla). By analogy to Grb2, an adaptor protein that couples receptor tyrosine kinases (RTK) to Ras, it is hypothesized that Dock links guidance receptors to the related Rho family GTPases, thereby modulating the actin cytoskeleton. While Drosophila guidance receptors linked to Dock have not been identified, the SH2 domain of Nck can bind to the cytoplasmic regions of several mammalian guidance receptors upon ligand stimulation, including c-Met (Kochhar, 1996) and EphB1 (Stein, 1998). In addition, mammalian Nck binds through its SH3 domains to proteins such as Pak and Prk2, which in turn bind to and are activated by Rho family GTPases. As a step toward understanding how Dock regulates downstream effectors, the function in R cell axon guidance of a Drosophila homolog of one of these, Pak (p21-activated kinase) was examined (Hing, 1999 and references).
Dock and Drosophila PAK-kinase have been shown to physically interact as do mammalian NIK and PAK (Bokoch, 1996 and Lu, 1997). If Dock and Pak function in the same signaling pathway in developing R cells to regulate growth cone motility, they would be expected to colocalize in these cells. R cells extend their axons into the optic lobe during the third instar of larval development. Dock staining is markedly enriched in the lamina and medulla neuropils, consistent with its localization to R cell axons and growth cones (Garrity, 1996). In contrast, Dock is only expressed at low levels in the cell bodies of developing R cells as well as in neuronal cell bodies in the cortical regions in the lamina and medulla. Strong staining is seen, however, in the neuropils of the optic disc. The lamina and medulla neuropils contain no neuronal cell bodies and comprise axonal processes and growth cones. Both anti-Dock and anti-Pak antibodies stain the medulla neuropil uniformly, indicating that these proteins are expressed on many visual system fibers. Since the R7 and R8 axons only contribute a small fraction of the total number of fibers in the medulla, it is not possible to assess whether Pak and Dock are coexpressed in these axons. In contrast, at this stage in development, the vast majority of the processes in the lamina neuropil belong to R cells, including the expanded R1-R6 growth cones and axons of R7 and R8. Hence, Pak, like Dock, preferentially localizes to axons and growth cones (Hing, 1999).
To assess whether Pak is required for growth cone guidance, mutations disrupting its function were identified. It was assumed that, like dock, null mutations in Pak would cause recessive lethality. Accordingly, lethal mutations were identified in a small region of the chromosome within which Pak maps. From 9440 mutant lines containing randomly mutagenized third chromosomes, 238 lethal mutations mapping to a deficiency that deletes Pak were isolated. These were then tested against the same deficiency chromosome bearing a Pak-containing cosmid. The cosmid rescued 21 mutations. These fell into two groups based on complementation tests. A Pak cDNA expressed under the control of the heat shock promoter rescued the lethality associated with one complementation group, indicating that these mutations disrupt Pak function. These alleles are designated Pak1 to Pak13 (Hing, 1999).
To assess R cell projections in Pak mutants, eye-brain complexes from transheterozygous larvae were stained with the R cell-specific antibody mAb24B10. In wild type, R cell axons grow from the eye disc, through the optic stalk, and into the optic ganglia during the third instar of larval development. The eight R cell axons from each ommatidium form a single bundle. These bundles spread out upon entering the optic lobe and form a smooth topographic map that reflects the arrangement of ommatidia in the eye. Growth cones of R1-R6 are seen as a band of immunoreactivity. In contrast, individual R8 growth cones are readily observed in the medulla neuropil. They are evenly spaced and exhibit a characteristic expanded morphology. At this stage of development, few of the R7 axons stain with mAb24B10 (Hing, 1999).
In Pak strong loss-of-function mutants, R cell axons extend into the brain normally. However, these fibers do not spread evenly within the lamina and medulla. As a result, some regions are hyperinnervated while others lack innervation. In the medulla neuropil, R cell axons fail to find their proper targets but instead, terminate as thick, blunt-ended fascicles. Hence, in contrast to wild type, Pak mutant R cells do not elaborate a smooth topographic map in the lamina and medulla neuropils. A small fraction of the R2-R5 neurons project through the lamina and into the medulla, indicating a modest disruption in ganglion target specificity. Eye-specific expression of a wild-type Pak cDNA under the control of the GMR promoter rescues the mutant phenotype. These data indicate that Pak is required for axon targeting but that it is not required for axon outgrowth because in these mutants R cell axons extend in the correct direction and into the target region. The Pak phenotypes are essentially indistinguishable from those previously described (Garrity, 1996) in dock mutants. While Pak has a profound effect on R cell projections, it does not disrupt R cell fate determination or differentiation (Hing, 1999).
R cell axons not only target to specific regions of the developing optic ganglia, they play an essential role in inducing optic ganglion development. They induce the proliferation of neuronal precursor cells in the lamina, and subsequently they induce neuronal differentiation. R cell axons also induce lamina glial cell differentiation. These inductive processes were assessed in Pak mutants using BrdU labeling to detect proliferating lamina precursor cells, and anti-Elav and anti-Repo staining to assess lamina neuron and glial cell differentiation, respectively. These steps occur normally in Pak mutants. Hence, like dock, Pak is not required for lamina induction (Hing, 1999).
Mutation analysis of Pak reveals that the kinase domain, the Crib domain (which interacts with Rac/Cdc4), and the Dock-binding site are all required for Pak function in R cell axon guidance. To assess whether Pak acts downstream of Dock, a dominant gain-of-function form of Pak was constructed. Previous studies have demonstrated that membrane localization of human Pak1 to the membrane leads to kinase activation. Accordingly, a membrane-anchored version of Pak was constructed by fusing a myristylation signal from Drosophila Src1 to the N terminus of Pak and expressed in developing R cells. This construct is designated GMR-Pakmyr. A single copy of GMR-Pakmyr rescues the Pak mutant phenotype, indicating that Pakmyr retains wild-type activity. GMR-Pakmyr shows a dose-dependent dominant phenotype in R cells. Wild-type larvae carrying a single copy of GMR-Pakmyr show mild clumping of axons in the lamina cortex. The R8 projections in the medulla and the pattern of R cells in the eye disc, however, are normal. This defect is slightly enhanced in animals with two copies of GMR-Pakmyr. In larvae carrying four copies of GMR-Pakmyr, the R cell projection pattern is severely disrupted. In addition, the pattern of R cell clusters in the eye disc is highly abnormal. Remarkably, in these animals R cells delaminate from the eye disc epithelium and migrate through the optic stalk into the brain. Both membrane localization and kinase activity are required to induce motility, since larvae carrying four copies of either GMR-Pakmyr K459A (kinase inactive) or GMR-Pakwt (not membrane tethered) are indistinguishable from wild type. While kinase activity is necessary to induce guidance defects and cell motility, it is not sufficient. The R cell projection pattern in larvae carrying four copies of GMR-PakL115F, which encodes a constitutively active but soluble form of Pak, was also indistinguishable from wild type. In summary, the ability of GMR-Pakmyr to rescue a Pak mutant phenotype and to confer a dose-dependent dominant phenotype is consistent with it acting as a dominant gain-of-function allele (Hing, 1999).
If Pak functions downstream of Dock, then the constitutively active form of Pak should rescue Dock mutations. If a key step in Dock function is to recruit Pak to the membrane, then membrane-tethered Pak, GMR-Pakmyr, may rescue some aspects of the dock mutant phenotype. To test this, a single copy of the transgene was introduced into a dock null background. In dock mutants, R cell axons form large abnormal fascicles in the optic ganglia. This leads to hyperinnervated regions separated by areas lacking innervation in both the lamina and medulla. In addition to disrupting targeting, R cell axon terminals in the medulla are thick and blunt ended. The dock phenotype was shown to be substantially rescued by GMR-Pakmyr. Axon bundles between the lamina and medulla are thinner in rescued flies. Growth cones in the medulla are expanded and spread out more evenly, giving rise to an array of terminals. Quantification of the expanded growth cones in the medulla shows an increase from less than 2 in dock mutants to 64 in dock mutants carrying a copy of GMR-Pakmyr. This represents a restoration of about half the number of growth cones, compared with wild-type preparations of similar age. Rescue requires both myristylation and kinase activity; rescue is not observed with GMR-Pakwt, kinase inactive or constitutively active soluble Pak. These data are consistent with models in which recruitment of Pak to the membrane by Dock is an essential regulatory step in R cell axon guidance (Hing, 1999).
While these studies demonstrate that Pak is a primary target of Dock in R cell growth cones, little is known about the upstream signals that regulate Dock and Pak function. It is proposed that guidance signals, functioning through membrane bound receptors, recruit Dock and Pak to the membrane. Two observations support this view: (1) overexpression of a constitutively membrane-tethered form of Pak (Pakmyr) leads to marked changes in growth cone and cell motility, and (2) Pakmyr expressed at a lower level substantially rescues the Dock null mutant phenotype. The latter result is somewhat surprising, since it was envisioned that precise spatial regulation of Pak within the membrane by recruitment to activated guidance receptors is crucial to its guidance function, an activity that is unlikely to be replicated entirely by the myristylation tag. Activated guidance receptors may, in addition to recruiting Dock, lead to increased levels of phosphoinositides, which in turn provide an alternate route to recruit Pak/Pix into complexes induced by the activated guidance receptors. Alternatively, guidance signals may independently activate Cdc42 (or Rac) in specific spatial domains within the growth cone. These proteins may subsequently recruit Pakmyr to these regions through the CRIB domain. Although it is envisioned that recruitment of Pak to specific spatial domains in growth cone membranes is important in axon guidance, the possibility that general localization of Pak to the membrane is sufficient to promote its function in guidance cannot be ruled out (Hing, 1999).
The importance of regulating Pak activity in cells is underscored by the observations that misexpression of high levels of Pakmyr, beyond that necessary to rescue the dock mutant phenotype, induces these cells to delaminate from the eye disc epithelium and migrate into the brain. The recruitment of Pak and Dock to the membrane may nucleate formation of large signaling complexes. In addition to the potential link of Pak to tyrosine kinase signaling pathways through Dock, Pak also contains an extreme C-terminal domain that binds to the Gß subunit of trimeric G proteins. Indeed, the yeast Pak Ste20 is activated through this site in response to G protein signaling pathways stimulated by mating pheromone (Leeuw, 1998). Pix not only contains a Pak interaction site and a guanine nucleotide exchange factor activity, it also contains a PH domain that potentially links Pak to yet additional pathways, including those regulated by phosphoinositides. And finally, Dock also interacts with multiple proteins, including phosphatases (for example, Drosophila PTP61F; Clemens, 1996) and kinases, as well as other cytoskeletal regulators (e.g., N-WASP; McCarty, 1998). While a Dock/Pak complex is well suited to integrate multiple signals and transmit them to the changes in the actin-based cytoskeleton, Dock and Pak also may act separately in different combinations to regulate growth cone guidance (Hing, 1999).
In response to environmental and physiological changes, the synapse manifests plasticity while simultaneously maintains homeostasis. This study analyzed mutant synapses of henji, also known as diablo (dbo), at the Drosophila neuromuscular junction (NMJ). In henji mutants, NMJ growth is defective with appearance of satellite boutons. Transmission electron microscopy analysis indicates that the synaptic membrane region is expanded. The postsynaptic density (PSD) houses glutamate receptors GluRIIA and GluRIIB, which have distinct transmission properties. In henji mutants, GluRIIA abundance is upregulated but of GluRIIB is not. Electrophysiological results also support a GluR compositional shift towards a higher IIA/IIB ratio at henji NMJs. Strikingly, dPAK, a positive regulator for GluRIIA synaptic localization, accumulates at the henji PSD. Reducing the dpak gene dosage suppresses satellite boutons and GluRIIA accumulation at henji NMJs. In addition, dPAK associated with Henji through the Kelch repeats which is the domain essential for Henji localization and function at postsynapses. It is proposed that Henji acts at postsynapses to restrict both presynaptic bouton growth and postsynaptic GluRIIA abundance by modulating dPAK (Wang, 2016).-
Coordinated action and communication between pre- and postsynapses are essential in maintaining synaptic strength and plasticity. Presynaptic strength or release probability of synaptic vesicles involves layers of regulation including vesicle docking, fusion, and recycling, as well as endocytosis and exocytosis. Also, how postsynapses interpret the signal strength from presynapses depends largely on the abundance of neurotransmitter receptors at the synaptic membrane. During long-term potentiation, lateral diffusion of extrasynaptic AMPA receptor to synaptic sites is accelerated and the exocytosis of AMPAR is enhanced near the postsynaptic density (PSD), causing an accumulation of synaptic receptors. In contrast, under the long-term depression condition, synaptic AMPAR is reduced by hastened endocytosis. While molecular mechanisms are proposed to play roles in regulating and fine-tuning postsynaptic glutamate receptor (GluR) abundance in plasticity models, the developmental regulation of GluR abundance at the synaptic surface still needs to be elucidated. Synapses at the Drosophila neuromuscular junction (NMJ) use glutamate as the neurotransmitter, and have properties reminiscent of mammalian central excitatory synapses. Homologous to vertebrate AMPAR and kainate receptors, Drosophila GluR subunits assemble as tetramers to gate ion influx. Each functional receptor contains essential subunits (GluRIIC, GluRIID and GluRIIE) and either GluRIIA or GluRIIB; therefore, synaptic GluRs can be classified according to their subunit compositions as either A- or B-type receptors. These two types of receptors exhibit distinct developmental and functional properties. Newly-formed PSDs tend to accumulate more GluRIIA channels, while the IIA/IIB ratio becomes more balanced when PSDs mature. In addition, GluRIIB channels have much faster desensitization kinetics, which results in smaller quantal size than GluRIIA channels. Therefore, the synaptic composition of these two types of GluRs greatly influences the postsynaptic interpretation of neuronal activities. The Drosophila homolog of p21-activated kinase (dPAK) regulates GluRIIA abundance at the PSD; GluRIIA receptor clusters at the postsynaptic membrane are strongly reduced in dpak mutants. However, overexpression of dPAK in postsynapses is not sufficient to increase GluRIIA cluster size, suggesting that dPAK activity in regulating GluRIIA abundance is tightly controlled (Wang, 2016).
Ubiquitination and deubiquitination play critical roles in regulating synaptic functions. In loss-of-function mutants for highwire, a gene encoding a conserved E3 ubiquitin ligase, NMJs overgrow, producing supernumerary synaptic boutons. This phenotype is duplicated by overexpression of the deubiquitinating enzyme Fat facets (Faf) in presynapses. These studies underline the importance of balanced ubiquitination in synapse formation and function. Cullin-RING ubiquitin ligases (CRLs) are large protein complexes that confer substrate ubiquitination. Importantly, CRLs promote ubiquitination through substrate receptors that provide specific recognition of substrates for ubiquitination. The BTB-Kelch proteins are suggested to be the substrate receptors for Cul3-scaffolded CRLs. This study identified a BTB-Kelch-containing protein, Henji, also known as Dbo, which regulates NMJ growth and synaptic activity by restricting the clustering of GluRIIA. Synaptic size of henji mutants was significantly expanded, as viewed under transmission electron microscopy (TEM). Immunostaining for dPAK and GluRIIA also suggests larger areas of PSDs in the absence of Henji, and the intensity of each fluorescent punctum becomes stronger, indicating abnormal accumulation of these PSD proteins. By genetically reducing one gene dosage of dpak in henji mutants, GluRIIA accumulation and abnormal bouton morphology was suppressed. In contrast, reducing the gluriia gene dosage in henji mutants restored bouton morphology but failed to suppress dPAK accumulation. Thus, Henji regulates bouton morphology and GluRIIA clustering levels likely through a control of dPAK. Interestingly, while overexpression of dPAK, either constitutively active or dominantly negative, had no effects on GluRIIA clustering, overexpression of these dPAK forms in henji mutants modulated GluRIIA levels, indicating that Henji limits the action of dPAK to regulate GluRIIA synaptic abundance. Henji localized to the subsynaptic reticulum (SSR) surrounding synaptic sites, consistent with the idea that Henji functions as a gatekeeper for synaptic GluRIIA abundance (Wang, 2016).
This study shows that Henji functions at the postsynapse to regulate synaptic development and function at the NMJ. The PSD area is expanded and GluRIIA clusters abnormally accumulate at the PSD. Genetic evidences are provided to support that the elevation of GluRIIA synaptic abundance is at least partially caused by a corresponding accumulation of dPAK in henji mutants. Henji is sufficient to downregulate dPAK and GluRIIA levels and the Kelch repeats of Henji play the most critical role in this process. Henji tightly gates dPAK in regulating GluRIIA abundance, as dPAK enhances GluRIIA cluster abundance only when Henji is absent. Therefore, this study has identified a specific negative regulation of dPAK at the postsynaptic sites that contributes to the PSD formation and GluR cluster formation at the NMJ (Wang, 2016).
PAK proteins transduce various signaling activities to impinge on cytoskeleton dynamics. Through kinase activity-dependent and -independent mechanisms, PAK regulates not only actin- and microtubule-based cytoskeletal rearrangement but also the activity of motors acting on these cytoskeletal tracks. In mammalian systems, PAKs participate in many synaptic events including dendrite morphogenesis, neurotransmitter receptor trafficking, synaptic strength modulation, and activity-dependent plasticity. Pathologically, PAK dysregulation also contributes to serious neurodegenerative diseases, Huntington's disease and X-linked mental retardation (Wang, 2016).
At Drosophila NMJs, dPAK has divergent functions; loss of dpak causes a dramatic reduction in both Dlg and GluRIIA synaptic abundance, but the underlying molecular mechanisms have not been revealed. The current data show that Henji functions to restrict GluRIIA clustering but has no effect on Dlg levels, suggesting that Henji regulates one aspect of dPAK activities, probably via the SH2/SH3 adaptor protein Dock. Alternatively, Henji may function to limit dPAK protein levels locally near the postsynaptic region, rendering its influence on GluRIIA clustering, while dPAK that regulates Dlg may localize outside of the Henji-enriched region. Supporting this idea, Henji is specifically enriched around the SSR region instead of dispersed throughout the muscle cytosol. Moreover, ectopic Myc-dPAK localized at the postsynapse only when henji was mutated, indicating that Henji regulates dPAK postsynaptic localization (Wang, 2016).
The interaction with Rac, Cdc42, or both triggers autophosphorylation and subsequent conformational changes of PAK, resulting in kinase activation. The myristoylated dPAK that has been shown to be active in growth cones failed to enhance GluRIIA abundance at the NMJ. This result shows that dPAK is necessary to regulate GluRIIA synaptic abundance, but is itself tightly regulated at the synaptic protein level or the kinase activity. Indeed, evidence is provided to show specific negative regulation of dPAK by Henji; overexpression of dPAK CA that could not enhance GluRIIA abundance in WT larvae further increased the already enhanced GluRIIA levels in the henji mutant. Similar to the CA form, the DN form also showed no effect on GluRIIA when simply overexpressed in the WT background, but exhibited strong suppression of GluRIIA in the henji mutant background. Thus, regardless of the possible conformational differences between the CA and DN forms, Henji appears to confer a constitutive negative regulation of dPAK at postsynapses, suggesting a tight control that could be at subcellular localization. In contrast to CA and DN forms, activation of dPAK requires binding to Rac1 and Cdc42, and subsequent protein phosphorylation. This additional layer of regulation may serve as a limiting factor rendering dPAK WT from recruiting GluRIIA to PSDs regardless in WT or henji mutant background (Wang, 2016).
The structural feature suggests that Henji could function as a conventional substrate receptor of the Cul3-based E3 ligase complex. At Drosophila wing discs, Dbo functions as a Cul3-based E3 ligase to promote Dishevelled (Dsh) downregulation. Similar to the henji alleles, it was confirmed that the dbo [Δ25.1] allele and dbo RNAi were competent to induce dPAK and GluRIIA accumulation at the postsynapse. An immunoprecipitation experiment detected Henji and dPAK in the same complex, and dPAK also forms a complex with the C-terminal substrate-binding Kelch-repeats region. However, no notable or consistent increase was detected in Henji-dependent dPAK poly-ubiquitination in both S2 cells and larval extracts. Also, the Cul3-binding BTB domain of Henji seems dispensable in the suppression of dPAK levels in henji mutants. Importantly, Cul3 knockdown in muscle cells failed to cause any accumulation of GluRIIA and dPAK at the NMJ. Sensitive genetic interaction between henji and Cul3 failed to induced dPAK and GluRIIA accumulation. Dbo functions together with another BTB-Kelch protein Kelch (Kel) to downregulate Dsh. However, Kel negatively regulates GluRIIA levels without affecting dPAK localization at the postsynaptic site. This data argues that Kel functions in a distinct pathway to Henji in postsynaptic regulation of GluRIIA. Taken together, no direct evidence was found to support that dPAK is downregulated by Henji through ubiquitination-dependent degradation. Alternately, Henji could bind dPAK near the postsynaptic region and this interaction may block the recruitment or localization of dPAK onto postsynaptic sites. Under this model, dPAK is less restricted and has a higher propensity to localize at postsynaptic sites in the absence of Henji, resulting in synaptic accumulation of dPAK and GluRIIA expansions (Wang, 2016).
As many synaptic events require rapid responses, local regulation of protein levels becomes crucial in synapses. To achieve accurate modulation, certain synaptic proteins should be selectively controlled under different developmental or environmental contexts. Indeed, emerging evidence shows that various aspects of synapse formation and function are under the control of the ubiquitin proteasome system (UPS), including synapse formation, morphogenesis, synaptic pruning and elimination, neurotransmission, and activity-dependent plasticity. In particular, the membrane abundance of postsynaptic GluR that modulates synaptic function can be regulated by components of the UPS. When Apc2, the gene encoding Drosophila APC/C E3 ligase, is mutated, GluRIIA shows excess accumulation but the molecular mechanism was not elucidated. Similarly, loss of the substrate adaptor BTB-Kelch protein KEL-8 in C. elegans also results in the stabilization of GLR-1-ubiquitin conjugates. However, no evidence shows direct ubiquitination and degradation of GLR-1 by KEL-8. Also, absence of the LIN-23-APC/C complex in C. elegans affects GLR-1 abundance at postsynaptic sites without altering the level of ubiquitinated GLR-1. Therefore, GLR-1 receptor endocytosis and recycling or ubiquitination and degradation of GLR-1-associated scaffold proteins are proposed to be the underlying mechanism for E3 ligase regulation. In mammals, endocytosis of AMPAR can be influenced by poly-ubiquitination and degradation of the prominent postsynaptic scaffold protein PSD-95 (Wang, 2016).
This study describes a novel regulation by the BTB-Kelch protein Henji on synaptic GluRIIA levels. By limiting GluRIIA synaptic levels, Henji modulates the postsynaptic output in response to presynaptic glutamate release. In the absence of Henji, quantal size is elevated, coinciding with an increase in the postsynaptic GluRIIA/GluRIIB ratio. In a previous study, increases in the GluRIIA/GluRIIB ratio by overexpressing a GluRIIA transgene in the muscle or by reducing the gene copy of gluriib promote NMJ growth, but co-expression of both GluRIIA and GluRIIB did not alter the bouton number. Combined with the current findings, those data provide a link between an increased GluRIIA-mediated postsynaptic response and bouton addition at NMJs. However, satellite boutons were not detected following GluRIIA overexpression. One possibility is that satellite boutons are considered as immature boutons and their appearance may indicate the tendency for NMJ expansion, as in the case of excess BMP signaling. Failure to become mature boutons may be caused by the lack of cooperation with other factors such as components of the presynaptic endocytic pathway, actin cytoskeleton rearrangement or neuronal activity. No significant alterations in endocytosis and the BMP pathway in the henji mutant. Nevertheless, it cannot be ruled out that Henji may modulate other presynaptic events that are defective in henji mutants to interfere with bouton maturation (Wang, 2016).
Bases in 5' UTR - 268
Bases in 3' UTR - 783
Two blocks of highly conserved sequences have been identified in PAK-kinase of Drosophila: the serine/threonine kinase domain and a stretch of amino acids identified as the p21-binding domain. The p21-binding domain serves to bind to small GTP-binding proteins such as Rac and Cdc42. The p21-binding domain is most homologous to that of the original rat PAK sequence and to domains from C. elegans and human PAK-Ste20 family members with homologies on the order of 80%. There is considerably less homology to the corresponding domains of yeast Ste20 (Harden, 1996).
date revised: 5 August 2021
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