par-1: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - par-1

Synonyms -

Cytological map position - 56D

Function - signaling

Keywords - anterior/posterior polarity, cytoskeleton

Symbol - par-1

FlyBase ID: FBgn0260934

Genetic map position -

Classification - par-1 serine/threonine kinase

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | Entrez Gene | UniGene

Recent literature
Ando, K., Maruko-Otake, A., Ohtake, Y., Hayashishita, M., Sekiya, M. and Iijima, K. M. (2016). Stabilization of microtubule-unbound Tau via Tau phosphorylation at Ser262/356 by Par-1/MARK contributes to augmentation of AD-related phosphorylation and Aβ42-induced Tau toxicity. PLoS Genet 12: e1005917. PubMed ID: 27023670
Summary:
To prevent the cascade of events leading to neurodegeneration in Alzheimer's disease (AD), it is essential to elucidate the mechanisms underlying the initial events of tau mismetabolism. In this study, using transgenic Drosophila co-expressing human tau and Aβ, tau phosphorylation at AD-related Ser262/356 stabilized microtubule-unbound tau was found in the early phase of tau mismetabolism, leading to neurodegeneration. Aβ increased the level of tau detached from microtubules, independent of the phosphorylation status at GSK3-targeted SP/TP sites. Such mislocalized tau proteins, especially the less phosphorylated species, were stabilized by phosphorylation at Ser262/356 via PAR-1/MARK. Levels of Ser262 phosphorylation were increased by Aβ42, and blocking this stabilization of tau suppressed Aβ42-mediated augmentation of tau toxicity and an increase in the levels of tau phosphorylation at the SP/TP site Thr231, suggesting that this process may be involved in AD pathogenesis. In contrast to PAR-1/MARK, blocking tau phosphorylation at SP/TP sites by knockdown of Sgg/GSK3 did not reduce tau levels, suppress tau mislocalization to the cytosol, or diminish Aβ-mediated augmentation of tau toxicity. These results suggest that stabilization of microtubule-unbound tau by phosphorylation at Ser262/356 via the PAR-1/MARK may act in the initial steps of tau mismetabolism in AD pathogenesis, and that such tau species may represent a potential therapeutic target for AD.
Barber, K.R., Tanquary, J., Bush, K., Shaw, A., Woodson, M., Sherman, M. and Wairkar, Y.P. (2017). Active zone proteins are transported via distinct mechanisms regulated by Par-1 kinase. PLoS Genet 13: e1006621. PubMed ID: 28222093
Summary:
Disruption of synapses underlies a plethora of neurodevelopmental and neurodegenerative disease. Presynaptic specialization called the active zone plays a critical role in the communication with postsynaptic neuron. While the role of many proteins at the active zones in synaptic communication is relatively well studied, very little is known about how these proteins are transported to the synapses. For example, are there distinct mechanisms for the transport of active zone components or are they all transported in the same transport vesicle? Is active zone protein transport regulated? This study shows that overexpression of Par-1/MARK kinase, a protein whose misregulation has been implicated in Autism spectrum disorders (ASDs) and neurodegenerative disorders, lead to a specific block in the transport of an active zone protein component- Bruchpilot at Drosophila neuromuscular junctions. Consistent with a block in axonal transport, there is a decrease in number of active zones and reduced neurotransmission in flies overexpressing Par-1 kinase. Interestingly, Par-1 was found to act independently of Tau-one of the most well studied substrates of Par-1, revealing a presynaptic function for Par-1 that is independent of Tau. Thus, this study strongly suggests that there are distinct mechanisms that transport components of active zones and that they are tightly regulated.
Herzmann, S., Krumkamp, R., Rode, S., Kintrup, C. and Rumpf, S. (2017). PAR-1 promotes microtubule breakdown during dendrite pruning in Drosophila. EMBO J 36(13): 1981-1991. PubMed ID: 28554895
Summary:
Pruning of unspecific neurites is an important mechanism during neuronal morphogenesis. Drosophila sensory neurons prune their dendrites during metamorphosis. Pruning dendrites are severed in their proximal regions. Prior to severing, dendritic microtubules undergo local disassembly, and dendrites thin extensively through local endocytosis. Microtubule disassembly requires a katanin homologue, but the signals initiating microtubule breakdown are not known. This study shows that the kinase PAR-1 is required for pruning and dendritic microtubule breakdown. The data show that neurons lacking PAR-1 fail to break down dendritic microtubules, and PAR-1 is required for an increase in neuronal microtubule dynamics at the onset of metamorphosis. Mammalian PAR-1 is a known Tau kinase, and genetic interactions suggest that PAR-1 promotes microtubule breakdown largely via inhibition of Tau also in Drosophila. Finally, PAR-1 is also required for dendritic thinning, suggesting that microtubule breakdown might precede ensuing plasma membrane alterations. These results shed light on the signaling cascades and epistatic relationships involved in neurite destabilization during dendrite pruning.
BIOLOGICAL OVERVIEW

Par-1 family members share a conserved function in the generation of cell polarity (see Nelson, 1997 for review). Drosophila par-1 mutants show a novel polarity phenotype in which Bicoid mRNA accumulates normally at the anterior, but Oskar mRNA is redirected to the center of the oocyte, resulting in embryonic patterning defects. These phenotypes arise from a disorganization of the oocyte microtubule cytoskeleton (Shulman, 2000).

Homologs of Par-1 participate in cell polarization in organisms as diverse as yeast, C. elegans and mammals. In the C. elegans oocyte, unlike in the Drosophila oocyte, there is no predetermined A/P polarity, and the axis is polarized instead by sperm entry, which defines the posterior pole. This event triggers a rearrangement of the cortical actin cytoskeleton in which actin foci migrate toward the anterior pole, generating cytoplasmic flows in the cell interior that move the P granules to the posterior. The cell then divides asymmetrically to generate a large anterior AB cell and a smaller posterior P1 cell, which inherits the P granules and subsequently gives rise to the germline lineage. Consistent with this actin-based mechanism for cell polarization, actin depolymerizing drugs block the partitioning of the cytoplasmic determinates known as P granules and cause a symmetric division, whereas microtubule depolymerization has no effect on these processes. In addition to actin, a number of genes have been shown to play a role in the establishment of A/P polarity in C. elegans, including the maternal-effect gene par-1. Mutations in par-1 cause a symmetric first division and block the segregation of the P granules and other determinants along the A/P axis. The PAR-1 serine/threonine kinase is itself asymmetrically localized to the posterior cortex of the one-cell zygote and segregated into P1 after the first division (Guo, 1995). The localization of PAR-1 is dependent upon other par gene products, including PAR-2 and PAR-3 (Drosophila homolog: Bazooka), and also requires the actin cytoskeleton and the nonmuscle myosin, NMY-2, which interacts directly with a C-terminal region of PAR-1 (Etemad-Moghadam, 1995; Boyd, 1996; Guo, 1996).

Disruption of an Schizosaccharomyces pombe par-1 homolog, kin1, causes cells to lose their normal rod-like shape and grow as spheres (Levin, 1990). Mammalian PAR-1 homologs localize to the lateral membrane domain of cultured epithelial cells, and dominant-negative versions of these proteins disrupt apical-basal polarity (Bohm, 1997). The first mammalian homologs of PAR-1, the MARKs, were identified as kinases that phosphorylate the microtubule-associated proteins: Tau, MAP2, and MAP4 (Drewes, 1995, Drewes, 1997; Illenberger, 1996). MARK-induced phosphorylation disrupts MAP binding to microtubules in vivo and leads to the destabilization of the microtubule cytoskeleton without affecting the organization of actin (Drewes, 1997; Ebneth, 1999). Drosophila and mammalian homologs of PAR-1 may therefore have a different function from the C. elegans kinase, since the Drosophila and mammalian proteins regulate microtubule dynamics, while the PAR-1-dependent polarization of the A/P axis in C. elegans is apparently microtubule-independent (Shulman, 2000 and references therein).

In Drosophila, the anterior-posterior (A/P) axis becomes polarized very early in oogenesis, when the oocyte moves to the posterior of the germline cyst. Here, the oocyte signals to the adjacent follicle cells, inducing them to adopt a posterior fate, and these cells subsequently send an unidentified signal back to the oocyte to establish A/P polarity. This signal induces the disassembly of a microtubule organizing center (MTOC) positioned at the oocyte posterior, and microtubules are nucleated from a new anterior MTOC to form an A/P gradient in which the minus ends appear to lie at the anterior pole and the plus ends at the posterior. This polarized microtubule network defines the A/P axis by directing the localization of Bicoid and Oskar mRNAs to opposite poles of the oocyte. The posterior localization of OSK mRNA is the key step in pole plasm assembly, since Osk protein nucleates the assembly of the polar granules, at least in part, by directly recruiting components such as Vasa. Mutations in genes that require OSK mRNA localization or the assembly of the polar granules have been shown to disrupt the recruitment of germline and posterior determinants to the posterior pole, resulting in a 'posterior group' phenotype in which embryos lack pole cells and abdominal segments. In Drosophila, mutations in par-1 disrupt the polarized organization of the oocyte microtubule network and block the posterior localization of OSK mRNA, leading to defects in the posterior patterning of the embryo and the formation of the germ cells (Shulman, 2000 and references therein).

Pole plasm formation depends on the stepwise recruitment of a number of posterior group gene products to the posterior pole. The posterior localization of Stauffen and Oskar mRNA leads to the translational activation of the latter to produce Osk protein, which then anchors the complex and recruits Vasa protein. To determine where PAR-1 lies in this hierarchy, its localization was examined in various posterior group mutants. Par-1 localization at the oocyte posterior is unaffected in vasPD egg chambers, and in homozygotes for osk missense mutations, in which OSK mRNA is localized and anchored at the posterior, but fail to recruit Vasa. In contrast, the strong osk nonsense allele, osk54, completely abolishes the posterior localization of Par-1 and null mutations in stau have a similar effect. Thus, the recruitment of PAR-1 to the posterior is upstream and independent of vasa, but requires osk and stau (Shulman, 2000).

Since the posterior localization of OSK mRNA, Stau, and Osk are interdependent, these experiments do not distinguish which of these components is responsible for recruiting PAR-1 to the posterior pole. Therefore use was made of an osk-bcd 3'-UTR transgene, in which the bcd localization signal directs the Stau-independent localization of OSK mRNA and protein to the anterior of the oocyte. In egg chambers expressing this construct, Par-1 localizes to the anterior as well as the posterior pole of the oocyte. Thus, Par-1 must interact directly or indirectly with either Osk protein or a region of OSK mRNA other than the 3'-UTR, which is absent from the transgene. Taken together, these results are most consistent with a model in which Par-1 associates with OSK mRNA, since both show a transient localization at the anterior of wild-type stage 9 oocytes, whereas Osk protein is not translated until the mRNA reaches the posterior (Shulman, 2000).

In C. elegans, the posterior localization of PAR-1 requires the activity of a number of genes, including par-3 and nmy-2 (Etemad-Moghadam, 1995; Guo, 1996). To determine whether Par-1 localization in Drosophila has any features in common with C. elegans, mutant germline clones of spaghetti-squashAX3, a null allele for the nonmuscle myosin II regulatory light chain, and bazooka4, a strong mutation in the par-3 homolog were generated. Neither of these mutations had an effect on Par-1 localization to the posterior, and there appear to be no other homologs of these genes in the Drosophila genome, indicating that the kinase is recruited to the posterior by distinct mechanisms in the two organisms (Shulman, 2000).

Given the lack of pole plasm in embryos derived from par-1 mothers, the earliest steps of pole plasm assembly during oogenesis were examined. In par-1 egg chambers, OSK mRNA localizes normally through stage 7, but then diverges strikingly from the wild-type pattern. In the strongest viable allelic combination, par-16323/par-1W3, OSK mRNA is never detected at the posterior, and is either mislocalized to an ectopic site in the center of the oocyte (73%) or not localized at all (27%). This unusual OSK mRNA 'dot' forms as early as stage 8, and can persist until stage 11, the latest stage that can be examined. Stau protein shows an identical mislocalization to the middle of the oocyte in these mutants. Weaker allelic combinations show a similar abnormal pattern of Stau and OSK mRNA localization, but with lower penetrance. In addition, these egg chambers often show an intermediate phenotype in which some OSK RNA forms a dot in the middle of the oocyte while the rest localizes normally to the posterior cortex. Occasional dots of mislocalized OSK mRNA are even observed in par-1W3 heterozygotes, indicating that par-1 has a slight dominant haplo-insufficient phenotype. Overall, the penetrance of the OSK mRNA mislocalization phenotype correlates well with that of abdominal defects for each allelic combination (Shulman, 2000).

The localization of OSK mRNA is microtubule-dependent, and several mutants that disrupt this process do so by altering the organization of the oocyte microtubule network. It was therefore examined whether microtubule organization and polarity are also disrupted in par-1 oocytes. In wild-type oocytes, the microtubules are organized in an A/P gradient at stages 7-9 that can be visualized using a Tau:GFP fusion protein. In addition, the polarity of the microtubules can be assayed by expressing microtubule motor proteins fused to beta-galactosidase (beta-gal). A beta-gal fusion to the plus-end-directed motor, Kinesin (Kin:beta-gal), localizes to the posterior of the oocyte during stages 9-10 like OSK mRNA, whereas a Nod:beta-gal fusion localizes to the anterior. In par-1 mutant oocytes, Tau:GFP labels microtubules uniformly around the cortex, including the posterior pole, where they are never seen in wild type. Moreover, like OSK mRNA and Stau, Kin:beta-gal is mislocalized to the center of the oocyte, although a small amount of residual staining is often seen at the posterior. In contrast, Nod:beta-gal shows a normal localization to the anterior in par-1 mutant oocytes, suggesting that the microtubules are still nucleated from this pole. Like the other aspects of the par-1 phenotype, the disruption of microtubule organization is completely penetrant in par-16323/par-1W3 egg chambers, but less so in the weaker allelic combinations (Shulman, 2000).

The abnormal arrangement of microtubules in par-1 is similar to that seen in cappuccino (capu), spire (spir), and chickadee mutants, which disrupt OSK mRNA localization by causing a premature rearrangement of microtubules into a cortical array. Upon careful comparison, however, the microtubules in par-1 appear more diffuse than the tight, parallel bundles of capu egg chambers. Furthermore, whereas mutations in capu or spir cause the premature initiation of cytoplasmic streaming, the cytoplasmic movements of par-1 oocytes are indistinguishable from wild type. The microtubule organization in par-1 mutants also differs from that seen in grk mutants, which fail to disassemble the posterior MTOC, resulting in a focus of microtubules at the posterior pole that is never seen in par-1 oocytes (Shulman, 2000).

Despite extensive molecular investigation and several large-scale genetic screens, no common components have previously been found to be required for A/P axis polarization in Drosophila and C. elegans. Indeed, the primary axes of these two organisms are specified by different cues, at different stages of development, and by mechanisms with distinct cytoskeletal requirements. Nevertheless, in both systems, the axis is polarized within a single cell by an extrinsic spatial cue that triggers cytoskeletal and cytoplasmic rearrangements; in each case, these events culminate in the posterior localization of germline determinants (Shulman, 2000).

In the nematode, mutations in par-1 disrupt the positioning of the mitotic spindle, leading to a symmetric first division, and block the segregation of the P granules and other determinants to the posterior daughter blastomere, resulting in disorganized embryos that lack germ cells. In Drosophila, mutations in par-1 disrupt the polarized organization of the oocyte microtubule network and block the posterior localization of OSK mRNA, leading to defects in the posterior patterning of the embryo and the formation of the germ cells. The functional analogy revealed by par-1 mutant phenotypes in Drosophila and C. elegans is reinforced by the finding that Par-1 protein localizes to the posterior pole at the time when the A/P axis is being specified in both organisms, even though the mechanisms of localization are distinct. In the nematode, PAR-1 localization to the posterior requires the activity of PAR-2, PAR-3, and NMY-2. In contrast, homologs of PAR-3 and the NMY-2 light chain are not required for the localization of Drosophila Par-1, and its recruitment to the posterior of the oocyte at stage 9 depends instead on OSK mRNA. Nevertheless, Par-1 is one of the first molecules to localize to the posterior in each system, and provides an example of a common mediator and molecular marker of A/P polarity in flies and worms (Shulman, 2000 and references therin).

This conserved requirement for par-1 is somewhat surprising given the apparently distinct mechanisms by which the A/P axes form in Drosophila and C. elegans. The localization of the polar granules in Drosophila depends on the microtubule-dependent transport of OSK mRNA to the posterior of the oocyte, and the results provide support for the direct role for Drosophila Par-1 in remodeling the oocyte microtubule cytoskeleton. In contrast, the completed C. elegans genome reveals no osk homolog, and P-granule segregation during the first cell cycle requires the actin cytoskeleton, but not microtubules. A possible resolution to this paradox is suggested by several recent results that reveal the existence of parallel pathways for localizing the P granules to the germ cell lineage of C. elegans. Mutations in par-2 and pod-1 and RNAi against nmy-2 and mlc-4 severely impair or eliminate the cytoplasmic flows that normally localize the P granules, yet these particles can still segregate to the posterior. Thus, the one-cell zygote must still possess A/P polarity that can direct P-granule segregation in the absence of cytoplasmic flows. Furthermore, par-1 mutants do not disrupt the actin reorganization or the cytoplasmic flows during the first cell cycle. Instead, the P granules disappear and then reappear two divisions later in all cells of the embryo. PAR-1 is therefore required for the stability of the P granules at the one-cell stage, and for an A/P polarity that is independent of cytoplasmic flows. It is interesting to note that subsequent to the first division, P granules continue to segregate within the P lineage in the absence of flows, and localize in a microtubule-dependent manner via an association with one spindle pole during anaphase. A similar mechanism has been described for P-granule segregation during the first cell cycle in some nematode species which, like Drosophila, polarize the A/P axis during oogenesis. Since almost nothing is known about the alternative localization pathway in C. elegans, the role of PAR-1 in this process might therefore be more similar to its function in Drosophila than first appears (Shulman, 2000 and references therin).

Although Drosophila par-1 mutations cause typical posterior group phenotypes in the embryo, they have a different effect from all previously identified mutations on the polarization of the oocyte at stage 9. Mutations in genes such as grk, Notch, and PKA disrupt signaling from the posterior follicle cells and cause a similar mislocalization of OSK mRNA and Kin:beta-gal to the center of the oocyte as seen in par-1. However, in contrast to these mutants, the posterior MTOC is disassembled normally in par-1 oocytes, and both BCD mRNA localization and nuclear migration are unaffected. The par-1 phenotype is also distinguishable from that caused by mutations in capu, spir, and chickadee. These mutants show tight microtubule bundles and a complete delocalization of OSK mRNA, whereas par-1 oocytes show comparatively diffuse microtubule arrays and mislocalize osk to an ectopic 'dot.' Furthermore, par-1 has no effect on cytoplasmic streaming, grk mRNA localization, or dorsal/ventral patterning of the egg shell and embryo, processes that are all disrupted by the capu-like mutations. Since the par-1 null allele blocks oocyte development before stage 6, the possibility that the phenotype of the strongest viable par-1 mutant combination reflects an incompletely penetrant grk- or capu-like phenotype cannot be ruled out. This seems highly unlikely, however, because in contrast to weak grk or capu mutants, par-16323/par-1W3 oocytes produce a completely penetrant disruption of microtubule organization and OSK mRNA localization, but do not affect bcd and grk mRNA localization, nuclear migration, or cytoplasmic streaming. Par-1 therefore seems to be required for a novel step in the A/P polarization of the oocyte that is necessary for osk but not BCD mRNA localization (Shulman, 2000 and references therin).

Microtubule stainings and the behavior of the Kin:beta-gal and Nod:beta-gal fusion proteins have led to a simple model for the microtubule organization in the stage 9 oocyte, in which the majority of the minus ends are nucleated from the anterior cortex, with the plus ends extending toward the posterior pole. Between stages 7 and 8, at least two events must take place in the oocyte in order to generate this polarized microtubule array: (1) the signal from the posterior follicle cells induces the disassembly of the MTOC at the posterior of the oocyte, and (2) a new MTOC is activated along the anterior cortex. Both of these events seem to occur normally in par-1 mutants, since BCD mRNA and Nod:beta-gal show a wild-type localization to the anterior of the oocyte, and there is no ectopic focus of microtubules at the posterior pole. Thus, the par-1 phenotype reveals that the polarization of the oocyte microtubule network requires more than just a simple switch from a posterior to an anterior MTOC. If Kin:beta-gal and Nod:beta-gal are reliable markers for the ends of microtubules, the plus ends appear to be abnormally focused on the middle of the oocyte in par-1 mutants, whereas the minus ends are unaffected. Thus, Par-1 activity may be required to regulate the plus ends of the microtubules so that they direct OSK mRNA transport to the posterior pole (Shulman, 2000).

The mammalian homologs of Par-1, the MARKs, were identified for their ability to phosphorylate a repeated motif in the microtubule binding domains of Tau, MAP2, and MAP4 (Drewes, 1997). Phosphorylation at these sites reduces the binding affinity of these MAPs for microtubules and induces microtubule depolymerization. Thus, it is attractive to propose that Drosophila PAR-1 has a similar activity, and functions by phosphorylating MAPs to modulate oocyte microtubule dynamics (Shulman, 2000 and references therin).

Par-1 localizes to the posterior with OSK mRNA during stage 9, but par-1 mutants disrupt the microtubule organization as early as stage 8, and as a consequence, neither OSK mRNA nor Stau protein localize to the posterior pole. Par-1 activity is therefore required for its own osk-dependent localization. Consistent with this, mislocalized Par-1 protein in the center of the oocyte has been occasionally observed in weak par-1 allelic combinations. No asymmetrically localized Par-1 has been detected in the oocyte during the stages when the microtubule reorganization takes place, but it is possible that Par-1 function is localized instead through the regulation of its activation. Indeed, the activity of the MARKs has been shown to depend on the phosphorylation of regulatory sites within the kinase domain, and these residues are conserved in Drosophila Par-1 (Shulman, 2000). Such regulation may also occur in C. elegans, since the posterior localization of PAR-1 is not essential for all of its functions in polarizing the first cell division. In par-2 mutants, for example, the P granules often segregate normally, even though PAR-1 protein is not localized to the posterior, whereas par-1 mutants completely abolish P-granule partitioning (Kemphues, 1988; Boyd, 1996).

The finding that the role of Par-1 in A/P axis formation precedes its osk-dependent localization to the posterior raises the question of whether Par-1 has any additional functions once it has reached the posterior pole. This question is not straightforward to answer, since it is not possible at present to rescue the OSK mRNA localization phenotype of par-1 mutants without also rescuing the posterior localization of Par-1 protein. One possibility is that this constitutes a positive feedback loop that reinforces an earlier function in the polarization of the oocyte by concentrating the protein where it is needed. Alternatively, localized Par-1 could play a direct role in the downstream steps of pole plasm assembly. In C. elegans, PAR-1 has been implicated in the translational activation of pal-1 mRNA in the P1 blastomere, where its activity is required to relieve translational repression mediated by the RNA binding protein MEX-3 (Bowerman, 1997). The association of Drosophila Par-1 with OSK mRNA therefore raises the possibility that it may function in a similar manner to relieve Bruno-dependent translational repression once osk reaches the posterior pole (Shulman, 2000).

Although the polarization of the oocyte microtubule cytoskeleton is most sensitive to reductions in Par-1 activity in Drosophila, this kinase is also required at other stages of development. In the germarium, Par-1 is localized to the fusome, and germline clones of the par-1 null allele block oogenesis during the previtellogenic stages. Par-1 must therefore have an earlier, essential role, perhaps in specifying asymmetry during the divisions that give rise to the germline cyst or during the subsequent determination of the oocyte. Furthermore, consistent with a previous investigation of mammalian Par-1 homologs in cultured epithelial cells, Par-1 is localized to the basolateral membrane of the mature follicular epithelium, suggesting the possibility of a conserved role in epithelial polarization. Finally, par-1 must have additional functions during the development of the zygote, since the null allele is homozygous lethal, and this probably explains why it was not identified in previous screens for maternal-effect mutations that affect embryonic patterning (Shulman, 2000).

In conclusion, the parallels between the localization and function of Par-1 homologs in Drosophila, C. elegans, and mammalian systems indicate that this kinase family shares a conserved function in the generation of cell polarity. Although its exact requirement is not known in any of these contexts, the analysis of the Drosophila par-1 phenotype, coupled with the activity of the mammalian MARKs, strongly suggest that PAR-1 plays a direct role in polarizing the microtubule cytoskeleton, and Drosophila should therefore provide a valuable system for investigating the in vivo activities of these kinases (Shulman, 2000).

PAR-1 kinase phosphorylates Dlg and regulates its postsynaptic targeting at the Drosophila neuromuscular junction

Targeting of synaptic molecules to their proper location is essential for synaptic differentiation and plasticity. PSD-95/Dlg proteins have been established as key components of the postsynapse. However, the molecular mechanisms regulating the synaptic targeting, assembly, and disassembly of PSD-95/Dlg are not well understood. This study shows that PAR-1 kinase, a conserved cell polarity regulator, is critically involved in controlling the postsynaptic localization of Dlg. PAR-1 is prominently localized at the Drosophila neuromuscular junction (NMJ). Loss of PAR-1 function leads to increased synapse formation and synaptic transmission, whereas overexpression of PAR-1 has the opposite effects. PAR-1 directly phosphorylates Dlg at a conserved site and negatively regulates its mobility and targeting to the postsynapse. The ability of a nonphosphorylatable Dlg to largely rescue PAR-1-induced synaptic defects supports the idea that Dlg is a major synaptic substrate of PAR-1. Control of Dlg synaptic targeting by PAR-1-mediated phosphorylation thus constitutes a critical event in synaptogenesis (Zhang, 2007).

Rearrangement of synaptic protein composition and structure is a fundamental mechanism governing synaptic plasticity. As organizers of the postsynapse, PSD-95/Dlg proteins have been intensively studied as substrates mediating synaptic plasticity. The signaling pathways that couple internal or external cues to the localization and function of PSD-95/Dlg are not well defined. This study has found that PAR-1 kinase plays a critical role in regulating the postsynaptic targeting of Dlg at the Drosophila NMJ. PAR-1 does so by phosphorylating Dlg at a Ser residue in the GUK domain. The conservation of this Ser residue in all members of the MAGUK proteins suggests that this phosphorylation event may represent a general mechanism by which the MAGUK proteins are regulated. This is the first time the PAR-1 family of Ser/Thr kinase has been shown to play an important role in synaptic development and function (Zhang, 2007).

PAR-1 directly phosphorylates Dlg, and overactivation of PAR-1 disrupts Dlg's postsynaptic targeting. The physiological function of PAR-1 in regulating Dlg synaptic targeting is supported by loss-of-function analysis, which indicates that phosphorylation by PAR-1 negatively regulates Dlg synaptic targeting. Consistent with this, in vivo FRAP analysis shows that the nonphosphorylatable DlgSA-GFP recovers much faster than DlgWT-GFP, and that the recovery of DlgWT-GFP is facilitated by PAR-1 loss-of-function, but impeded by PAR-1 overexpression. At first glance, it may seem somewhat counterintuitive that DlgSA-GFP, which accumulates to a greater degree at the synapse than DlgWT-GFP does, is replaced more quickly and to a greater extent that DlgWT-GFP. Since FRAP analysis suggested that the recovered Dlg comes primarily from Dlg protein reserved or newly synthesized in the muscle cytoplasm rather than from diffusion of Dlg protein from the neighboring synapses, the most likely explanation is that PAR-1-mediated phosphorylation regulates the transport of Dlg from the extrasynaptic to the synaptic regions. DlgSA-GFP may be transported more efficiently from the extrasynaptic region to the postsynapse. Upon reaching the postsynapse, DlgSA-GFP may also associate with the synaptic membrane more tightly (Zhang, 2007).

Previous studies have demonstrated that the GUK domain, in which the S797 residue is located, plays an important role in the trafficking and synaptic targeting of Dlg. The importance of the GUK domain in mediating Dlg function is also highlighted by the fact that many of the identified dlg mutations are clustered in this domain. Two types of protein-protein interactions involving the GUK domain have been detected: (1) intramolecular interaction with the SH3 domain, and (2) protein-protein interactions with GUK binding proteins, including an MT binding protein and a kinesin motor. Since MT and MT-based motor proteins provide a major driving force for protein and mRNA trafficking, it is possible that PAR-1-mediated phosphorylation may regulate Dlg interaction with the MT-based transport system (Zhang, 2007).

Morphological and electrophysiological rescue experiments strongly support that Dlg is a critical downstream target through which PAR-1 impacts synapse differentiation and function. However, the rescue of PAR-1 overexpression-induced defects by DlgSA-GFP is not complete, raising the possibility that other synaptic substrates are affected by PAR-1. It is also possible that some of the PAR-1 overexpression phenotypes are neomorphic. A possible neomorphic effect caused by the synaptic upregulation of a kinase has recently been described. None of the known postsynaptic markers, such as CaMKII, FasII, or GluRIIA has the KXGS motif, suggesting that they may not be PAR-1 targets. In other developmental contexts, PAR-1/MARK kinases phosphorylate a number of substrates. Whether any of these PAR-1 substrates function at the synapse awaits further investigation. The existence of other synaptic targets of PAR-1 could also explain why it was not possilbe to effectively rescue par-1 mutant phenotypes with the Dlg-GFP variants, although there are other possible explanations for this result. For example, phosphorylated Dlg may possess certain biological activity that cannot be provided by DlgSD-GFP. Even if some of the phenotypes caused by altered PAR-1 activities are mediated by other substrates, several lines of evidence indicate that the mislocalization of Dlg is a primary effect of PAR-1 phosphorylation of Dlg, rather than a secondary consequence of synaptic damages caused by PAR-1 action on some unknown target(s): (1) the phospho-mimetic DlgSD-GFP is mislocalized in a wild-type background, in the presence of normal synaptic structures; (2) another postsynaptic marker, GluRIIA, retains its predominant postsynaptic localization in a PAR-1 overexpression situation; (3) the subsynaptic reticulum loss and synaptic transmission defects caused by PAR-1 overexpression using DlgSA-GFP could largely be rescued; (4) in a condition where postsynaptic structure was maintained with exogenous DlgSA-GFP, endogenous Dlg was still mislocalized in the presence of overexpressed PAR-1 (Zhang, 2007).

Recent studies suggest that posttranslational modification plays a role in regulating the trafficking of PSD-95/Dlg. In mammalian central synapses, N-terminal palmitoylation is critical for the intracellular sorting, postsynaptic targeting, and surface expression of PSD-95. Cyclin-dependent kinase 5 (Cdk5) phosphorylates the N-terminal region of PSD-95, inhibiting its oligomerization, channel clustering activity, and possibly, synaptic localization. This study establishes PAR-1-mediated phosphorylation at the C-terminal GUK domain as a regulatory mechanism in the synaptic targeting of Dlg. In addition, two independent studies have been conducted to study the function of CaMKII at the Drosophila NMJ. However, divergent results were obtained on the effect of CaMKII on synaptic development and function, and it appears that further studies are needed to clarify the function of CaMKII at the Drosophila NMJ. Future studies of upstream signaling events in the regulation of PAR-1 at the synapse, especially those which potentially regulate the PAR-1-Dlg phosphorylation cascade, will provide new insights on molecular mechanisms that regulate synaptic differentiation and plasticity (Zhang, 2007).

It is interesting to note that in addition to postsynaptic defects, altering PAR-1 activity leads to profound defects in presynaptic development and function. This indicates that PAR-1 regulates the coordinated maturation of pre- and postsynaptic structures. PAR-1 could regulate the adhesion between the pre- and postsynaptic membranes or trans-synaptic signaling. Intriguingly, a previous study has revealed a presynaptic localization and function for Dlg in regulating neurotransmission. Since a fraction of PAR-1 is localized at the presynapse, it raises the possibility that PAR-1 may also play a role there. Further studies are needed to test whether PAR-1 may act through Dlg or other substrates at the presynapse to affect neurotransmission. Previous studies have also implicated BMP as a retrograde signal that modulates presynaptic development and function in response to postsynaptic alterations. It would be interesting to explore the relationship between PAR-1 and Dlg-mediated synaptic effects and BMP-mediated retrograde signaling (Zhang, 2007).

The current model predicts that PAR-1 overactivation causes Dlg hyperphosphorylation and delocalization from the synapse, producing certain Dlg loss-of-function effects. In contrast, loss of PAR-1 function has the opposite effect, causing Dlg overactivation phenotypes. Most of the phenotypes observed are consistent with this model. For example, at the morphological level, PAR-1 overexpression and Dlg inactivation both lead to SSR loss, whereas loss of PAR-1 and overexpression of Dlg promote SSR growth. At the electrophysiological level, PAR-1 overexpression or Dlg loss of function leads to reduced EJC amplitude, whereas loss of PAR-1 has the opposite effect. The genetic interaction between PAR-1 and Dlg is also consistent with an antagonistic effect of PAR-1 on Dlg. Some inconsistencies of certain PAR-1 overexpression phenotypes and previously published dlg mutant phenotypes are noted. For example, overexpression of PAR-1 in the postsynapse causes reductions in both active zone number and synaptic vesicle density, whereas quite variable phenotypes, ranging from no obvious structural alteration in the presynapse to reduction in synaptic vesicle density or increase in active zone number, have been described for different dlg mutant alleles. Similarly, in electrophysiological studies, a decrease was found in both mEJC and EJC amplitudes, but no significant change in quantal content, in both Mhc>PAR-1 animals and dlgX1-2 mutants. These neurotransmission phenotypes are different from those previously reported for dlg mutant alleles dlgm52 and dlgv59, in which EJC was increased, whereas mEJC was not changed. However, a recent study also reported features of reduced neurotransmission in dlgX1-2 mutant. It is therefore possible that different dlg mutant alleles may differentially affect synaptic function (Zhang, 2007).

Recent studies have revealed a tight correlation between synaptic dysfunction and the pathogenesis of neurodegenerative diseases and other neurological disorders. In AD in particular, synaptic dysfunction occurs decades before the onset of amyloid plaque and neurofibrillary tangle formation and discernable neuronal loss. Intriguingly, loss of PSD-95 protein has been observed in AD patients. It is conceivable that under disease conditions, an increase of PAR-1/MARK activity might occur in response to certain neurotoxic insults, leading to abnormal phosphorylation and delocalization of PSD-95 from the postsynapse, eventually leading to neuronal dysfunction and death. Further studies in human AD postmortem tissues and mouse AD models will test the potential role of PAR-1/MARK kinases in regulating PSD-95 function and disease pathogenesis (Zhang, 2007).


GENE STRUCTURE

The par-1 locus spans approximately 30 kb, and encodes at least five transcripts that arise from a choice of three promoters and alternative splicing at the 3' end. Consequently, transcripts from each promoter are predicted to encode protein isoforms with distinct N-terminal domains, which have been termed N1, N2, and N3, preceding a shared kinase domain. Following exon 14, alternative splicing can bring the open reading frame to a STOP or extend it by about 300 bp that encode a conserved domain shared by all reported PAR-1 homologs. In addition, several internal splicing differences have been identified, as well as alternative splicing which solely affects the 5'- or 3'-UTR sequences. The Drosophila par-1 locus is further complicated by the presence of a nested gene, mei-W68, which encodes a homolog of S. cerevisiae Spo11, and is required for the initiation of double-strand breaks during meiotic recombination. mei-W68 shares a promoter and 5'-UTR with the N1 class of par-1 transcripts, and the coding sequence falls entirely within the first par-1 intron (Shulman, 2000).


PROTEIN STRUCTURE

Amino Acids - Protein isoforms of 833-1130 amino acids are predicted

Structural Domains

Drosophila Par-1 is a well-conserved member of the PAR-1 family of serine/threonine kinases and is approximately equally homologous to C. elegans PAR-1 and the mammalian MARKs. All of these proteins have very similar kinase domains and C-terminal domains, and show a dispersed but lower overall homology in the linker region between these domains. As with other family members, however, the amino terminal domains of Drosophila Par-1 are not conserved, and show no similarity to one another or to any proteins in the database. There are two other related kinases, Kp78A and Kp78B, in the 'complete' Drosophila genome, but these are both more divergent than Par-1, and lack the C-terminal domain characteristic of this family (Shulman, 2000).


par-1: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 20 May 2000

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