A fly Cdk5 regulatory subunit (Dp35; GenBank accession number AF231134) was identified from the Drosophila expressed sequence tag (EST) database using DNA sequence of clones obtained by degenerate PCR followed by cDNA and genomic library screens. Dp35 encodes a ~52 kDa protein with 31%-40% overall identity to other p35 family members, and 55%-66% identity in its carboxy-terminal half, where the Cdk5-binding and activation domains reside. Dp35 also has the amino-terminal myristoylation motif, which is conserved in p35 family members, indicating that the cDNA is full length (Connell-Crowley, 2000).
Dp35 and Cdk5 associate and cooperate functionally in vitro and in vivo. Dp35 has been shown to bind Cdk5. In an in vitro glutathione-S-transferase (GST) pull-down assay, fly Cdk5 associates with GST-Dp35 but not with GST alone, as does a mutant Cdk5 (Cdk5K33A) that is predicted to be kinase inactive. Moreover, Myc-epitope-tagged Dp35 (Dp35-Myc) expressed in human 293T cells associates with endogenous human Cdk5 in vivo, but not with Cdks 1, 2, 4 or 6. Dp35 activates Cdk5. Anti-Myc antibody immunoprecipitates from human 293T cells containing Dp35-Myc and human Cdk5 phosphorylated histone H1. Furthermore, anti-FLAG antibody immunoprecipitates from human 293T cells containing fly FLAG-epitope-tagged Cdk5 (Cdk5-FLAG) exhibit H1 kinase activity in the presence, but not in the absence, of Dp35. In contrast, Cdk5K33A-FLAG is catalytically inactive, although it does associate with Dp35. Dp35 interacts genetically with Cdk5 in the Drosophila eye (Connell-Crowley, 2000).
Expression of one copy of Cdk5-FLAG in all cells of the eye using the GMR-GAL4 driver has no effect on eye surface morphology, whereas one copy of Dp35-Myc causes a slight rough eye phenotype, presumably by activation of endogenous, low-level Cdk5. In contrast, coexpression of Cdk5-FLAG with Dp35-Myc severely disrupts eye morphology. This synthetic phenotype requires Cdk5 kinase activity because coexpression with two copies of Cdk5K33A-FLAG suppresses rather than enhances the mild rough eye phenotype observed with two copies of Dp35-Myc. Thus, like their mammalian orthologs, fly Cdk5 is activated by Dp35, whereas Cdk5K33A appears to act as a dominant-negative mutant by titrating Dp35 and will be referred to below as Cdk5dn (Connell-Crowley, 2000).
Cyclin-dependent kinase 5 activator (Cdk5alpha), is an activator of Cdk5 kinase activity and its expression is restricted to neurons. The complex of Ckd5/Cdk5alpha is essential for neurite outgrowth during neuronal differentiation and possibly also for neuronal degeneration. Drosophila Cdk5alpha-like, dCdk5alpha, has been isolated and characterized. The gene encoding this molecule is localized in the Drosophila chromosome region of 31D1-31D2. The expression of this gene is differentially regulated with a very low level at earlier developmental stages and reaches the highest level in the adult. The C-terminal of this protein shares high homology with the mammalian Cdk5alpha molecule. Constitutive over-expression of dCdk5alpha in transgenic flies significantly prolongs their recovery time from a 5 minute O2 deprivation or anoxia in older flies (15 days). Recovery times are not prolonged when younger flies (4 days old) are deprived of O2. In addition, anoxia up-regulates the expression of this gene. Taken together, the results in this report and others provide a framework for genetically dissecting the functions of Cdk5alpha/Cdk5 complex in the CNS (Ma, 1999).
Multisite hyperphosphorylation of tau has been implicated in the pathogenesis of neurodegenerative diseases including Alzheimer's disease (AD). However, the phosphorylation events critical for tau toxicity and mechanisms regulating these events are largely unknown. Drosophila PAR-1 kinase is shown to initiate tau toxicity by triggering a temporally ordered phosphorylation process. PAR-1 directly phosphorylates tau at S262 and S356. This phosphorylation event is a prerequisite for the action of downstream kinases, including glycogen synthase kinase 3 (GSK-3) and cyclin-dependent kinase-5 (Cdk5), to phosphorylate several other sites and generate disease-associated phospho-epitopes. The initiator role of PAR-1 is further underscored by the fact that mutating PAR-1 phosphorylation sites causes a much greater reduction of overall tau phosphorylation and toxicity than mutating S202, one of the downstream sites whose phosphorylation depends on prior PAR-1 action. These findings begin to differentiate the effects of various phosphorylation events on tau toxicity and provide potential therapeutic targets (Nishimura, 2004).
Drosophila has established itself as a model system for studying human neurodegenerative disorders. Fly models of tauopathy have been created by expressing wild-type or FTDP-linked mutant forms of h-tau. Using such models and based largely on overexpression experiments, it has been shown that Shaggy (GSK-3) can promote neurofibrillary tangle (NFT) pathology in photoreceptor neurons (Jackson, 2002). Whether GSK-3 and NFT are necessary for tau-mediated neurodegeneration, however, remains uncertain. Other studies have shown that tau-mediated neurodegeneration could occur without NFT and that GSK-3ß-induced tau hyperphosphorylation in mice could correlate inversely with neuropathology (Nishimura, 2004 and references therein).
Critical testing for a functional role of phosphorylation in tau-mediated neuropathology will require identifying the physiological tau kinase and assessing the consequence of removing this kinase activity on the disease process. Through loss-of-function and overexpression genetic studies and biochemical analysis, it has been shown that PAR-1 is a physiological tau kinase that plays a central role in regulating tau phosphorylation and toxicity in Drosophila. PAR-1 is a Ser/Thr kinase originally identified in C. elegans for its role in regulating cell polarity and asymmetric cell division. PAR-1 homologs have been found in eukaryotes ranging from yeast to mammals and exert essential cellular and developmental functions. MARK kinase, the mammalian homolog of PAR-1, regulates MT dynamics, epithelial cell polarity, and neuronal differentiation. Drosophila PAR-1 plays important roles in MT organization, oocyte differentiation, anterior-posterior axis formation, and Wingless signaling. While analyzing the neuronal function of PAR-1, it was found that Drosophila PAR-1 is a physiological kinase for fly Tau and h-tau. Overexpression of PAR-1 leads to elevated tau phosphorylation and enhanced toxicity, whereas removing PAR-1 function or mutating PAR-1 phosphorylation sites in tau abolishes tau toxicity. Furthermore, an initiator role for PAR-1 has been uncovered in a multisite phosphorylation process that generates pathogenic forms of tau. In this process, phosphorylation by PAR-1 precedes and is obligatory for downstream phosphorylation events, including those carried out by GSK-3 and Cdk5, to generate toxic tau. Consistent with PAR-1 playing an initiator role in the process, mutating PAR-1 phosphorylation sites causes a much more dramatic reduction of overall tau phosphorylation and toxicity than mutating one of the downstream Cdk5/GSK-3 phosphorylation sites. These findings have important implications for understanding the biogenesis of pathogenic tau in neurons and for developing mechanism-based therapeutic strategies (Nishimura, 2004).
Recent transgenic animal studies have implicated two kinases, GSK-3 and Cdk5, in the phosphorylation of tau in vivo. Analyses of tau phosphorylation status in transgenic mice overexpressing GSK-3 or Cdk5 have detected increased phosphorylation at certain sites previously identified as their in vitro phosphorylation sites. For example, S202 and PHF-1 sites (S396 and S404) have been shown to be prominent Cdk5 and GSK-3 phosphorylation sites, respectively, and the two kinases may have overlapping specificity at these sites. Tests were performed to see whether these sites in h-tauM were also phosphorylated by the corresponding fly kinases. The activity of Cdk5 is regulated by its binding with neuron-specific activators. Overexpression of Drosophila P35 activator has been shown to elevate endogenous Cdk5 activity. In P35 and h-tauM coexpression flies, the level of phosphorylation at S202 recognized by CP13 antibody is elevated. In addition, phosphorylation at AT270 sites was also significantly increased. Phosphorylation at AT100, AT180, and PHF-1 sites was relatively unchanged. Thus, phosphorylation at S202 and T181 responds to changes in Cdk5 levels. The eye morphology of P35 and h-tauM coexpressing flies appearssimilar to that of flies expressing h-tauM alone, suggesting that elevated Cdk5 activity does not significantly enhance tau toxicity. Shaggy and h-tauM coexpression flies were analyzed next. Coexpression of Shaggy and h-tau results in enhanced eye degeneration phenotypes. In the coexpression flies, significantly increased tau phosphorylation was observed at PHF-1, CP13, AT180, and AT100 sites. It is concluded that these phospho-epitopes contain GSK-3 phosphorylation sites and that elevated phosphorylation at these sites enhances tau toxicity (Nishimura, 2004).
The fact that many of the above-tested phosphorylation sites for GSK-3 and Cdk5 kinases are affected in S2A suggests that phosphorylation by the two kinases is regulated by prior PAR-1 action. To test this idea further, the phosphorylation status of GSK-3 and Cdk5 phosphorylation sites was analyzed in PAR-1 and h-tauM coexpression flies. In addition to 12E8 sites, significant increase of phosphorylation was observed at CP13 and PHF-1 sites in these flies. In contrast, phosphorylation at other sites such as AT100 sites was little changed, suggesting that PAR-1 is not a rate-limiting factor for these phosphorylation events. Since in vitro kinase assays showed that PAR-1 is incapable of directly phosphorylating the CP13 and PHF-1 sites, the elevated phosphorylation at these sites in PAR-1 coexpressing flies are likely mediated by downstream kinases such as Cdk5 and GSK-3 (Nishimura, 2004).
Whether coexpression of PAR-1, GSK-3, or Cdk5 has any modulating effect on S2A toxicity was further tested in vivo. PAR-1 and S2A coexpression flies showed a mild rough eye phenotype similar to PAR-1 overexpression alone, indicating that PAR-1 overexpression does not confer additional toxicity to S2A. Co-overexpression of GSK-3 or Cdk-5 also did not change S2A toxicity. These results further support the notion that phosphorylation by PAR-1 at S262 and S356 is a prerequisite for the subsequent phosphorylation by downstream kinases such as GSK-3 and Cdk5 to generate toxic tau species (Nishimura, 2004).
Since the S2A mutation disrupts tau phosphorylation at multiple downstream sites, it does not allow distinguishing the contribution of individual phosphorylation sites to tau toxicity. This issue was addressed by making point mutations in the downstream phosphorylation sites. Focus was placed on the S202 site because it is phosphorylated by Cdk5 and GSK-3 in vivo and because AT8 antibody, which is sensitive to phosphorylation at this site, was considered an Alzheimer-diagnostic antibody. Transgenic flies were generated that express h-tauM containing an Ala substitution at S202 (S202A). Western blot analysis demonstrated that, as predicted, S202A protein was no longer recognized by CP13 or AT8 antibodies. Significantly, phosphorylation at 12E8, AT100, PHF-1, AT180, and AT270 sites was unaffected by S202A mutation. This suggests that unlike S262 and S356 sites, the phosphorylation state of S202 does not influence that of other sites. Examination of external eye morphology by SEM and photoreceptor staining of eye sections has shown that, unlike S2A, S202A is as toxic as h-tauM. This suggests that phosphorylation by GSK-3 and Cdk5 at S202 site plays a rather limited role in conferring tau toxicity. This result supports the notion that PAR-1 plays an initiator role in the pathogenic phosphorylation process and further suggests that phosphorylation at downstream sites other than S202 or a combination of those downstream phosphorylation events makes a major contribution to tau toxicity (Nishimura, 2004).
Thus PAR-1, the fly homolog of mammalian MARK kinase, plays a central role in conferring tau toxicity in vivo. This study reveals PAR-1 function in triggering a temporally ordered phosphorylation process that is responsible for generating toxic forms of tau. This multisite phosphorylation process involves downstream kinases such as Cdk5 and GSK-3, whose action depends on prior phosphorylation of h-tau by PAR-1. A nonphosphorylatable mutation at S202, one of the downstream GSK-3/Cdk5 target sites whose phosphorylation depends on prior PAR-1 action, has a much smaller impact on overall tau phosphorylation and toxicity than mutations at PAR-1 phosphorylating sites. This strongly supports the initiator role of PAR-1 in generating toxic species of tau and further implies that the toxic form of tau may be phosphorylated at a subset or all of the other downstream sites (Nishimura, 2004).
It was previously shown that PAR-1 regulates the Wingless/Wnt pathway in Drosophila and Xenopus by phosphorylating the core component Dishevelled. It is thus interesting that GSK-3, another core component of Wingless pathway, acts downstream of PAR-1 to phosphorylate h-tau. These results are consistent with the notion that the Wingless pathway may be involved in regulating tau phosphorylation. It has been proposed that the pathway components are utilized differently in tau phosphorylation than in canonical Wnt signaling. The data indicate that PAR-1 and GSK-3 directly phosphorylate tau in an ordered fashion, with PAR-1 action preceding that of GSK-3. One parsimonious explanation for the requirement of prior phosphorylation by PAR-1 is that PAR-1 phosphorylation reduces the affinity of tau for MT and releases it from the MT network, therefore allowing easy access by other kinases. If that is the case, the mechanism may operate in a region-specific manner since certain phosphorylation sites do not depend on prior PAR-1 action. The data are also consistent with the idea that PAR-1 phosphorylation at 12E8 sites provides docking sites for intermediary kinase(s) and/or adaptor molecule(s), which facilitate subsequent phosphorylation by GSK-3 and Cdk5. It appears that the phosphorylation at certain downstream sites is achieved through a complex process. For example, phosphorylation at AT100 sites depends on prior PAR-1 action, but PAR-1 co-overexpression does not increase phosphorylation at these sites. Instead, co-overexpression of GSK-3 can lead to increased phosphorylation at AT100 sites. Previous in vitro studies have shown that the generation of AT100 epitope requires a PHF-like conformation of tau and the sequential phosphorylation by GSK-3 and PKA. It remains to be determined whether GSK-3 and PKA act downstream of PAR-1 to phosphorylate AT100 sites in flies (Nishimura, 2004).
Although Abl functions in mature neurons, work to date has not addressed Abl's role on Cdk5 in neurodegeneration. β-amyloid (Aβ42) initiates Abl kinase activity and blockade of Abl kinase rescues both Drosophila and mammalian neuronal cells from cell death. Activated Abl kinase is necessary for the binding, activation, and translocalization of Cdk5 in Drosophila neuronal cells. Conversion of p35 into p25 is not observed in Aβ42-triggered Drosophila neurodegeneration, suggesting that Cdk5 activation and protein translocalization can be p25-independent. These genetic studies also showed that abl mutations repress Aβ42-induced Cdk5 activity and neurodegeneration in Drosophila eyes. Although Aβ42 induces conversion of p35 to p25 in mammalian cells, it does not sufficiently induce Cdk5 activation when c-Abl kinase activity is suppressed. Therefore, it is proposed that Abl and p35/p25 cooperate in promoting Cdk5-pY15, which deregulates Cdk5 activity and subcellular localization in Aβ42-triggered neurodegeneration (Lin, 2007).
Like Cdk5, cellular Abl functions in neural development and its kinase activity and subcellular localization are tightly regulated. This study shows that Abl appears to be essential for Aβ42-triggered Drosophila neurodegeneration both in vivo and in vitro. It is of interest in this regard that Abl may serve as a putative molecular target to stop the progress of neurodegeneration. Interestingly, the anti-leukemic agent Abl kinase inhibitor, STI571, has been shown to rescue the Aβ42-induced neurodegeneration in both Drosophila and mammalian cells. However, STI571 is probably not an ideal reagent for testing this idea in vivo because of its low penetration capability through the blood-brain barrier. Another previous link between Aβ42 and Abl inhibition by STI571 has been reported. Aβ42 production is reduced by STI571 in neuronal cultures and in guinea-pig brain. Therefore, it is reasonable to speculate that Abl kinases might affect amyloid signaling at various points including Aβ42 production (Lin, 2007).
The cyclin-dependent kinase Cdk5 has attracted a great deal of attention both because of its roles in cell migration and axon patterning, and the extensive data implicating it in adult-onset neurodegeneration in mammals. Both the kinase activity and the biological effects of Cdk5 are absolutely dependent on association with an activating subunit, called p35. Drosophila lacking the Cdk5 activator, D-p35, display a wide range of defects in embryonic axon patterning. While viable and fertile, p35 mutant adults display progressive, age-dependent loss of motor function and have a significantly shortened lifespan (Connell-Crowley, 2007).
Previous studies employing overexpression of dominant transgenic constructs suggested that reduction of Cdk5 activity leads to errors in axon guidance in vivo. The p35 null phenotype reported here supports those findings, confirming the necessary role of Cdk5 and p35 in embryonic axon guidance. Moreover, absence of p35 causes age-dependent deficits in motor function, as demonstrated by progressive degradation of motor function, culminating in periods of rigidity, and associated with premature death (Connell-Crowley, 2007).
The axon patterning defects observed in the p35 null mutant mimic closely, both in kind and in severity, the defects produced by neuronal expression of a kinase-inactive Cdk5. This suggests that the dominant negative Cdk5 was both faithful and effective in revealing the function of Cdk5/p35. Moreover, while it is clear from the Drosophila genome sequence and from molecular studies that there is only a single p35 family member in Drosophila, the observation that expression of the dominant-negative Cdk5 in the p35 null background does not yield a stronger phenotype than either the mutant or the transgene alone suggests that there is not likely to be another major Cdk5 activator protein of some other molecular nature in growing Drosophila axons (Connell-Crowley, 2007).
Careful observation of the p35 null stock suggested the presence of behavioral deficits and reduced adult vigor. Quantitative behavioral assays supported this impression, revealing that while motor function in the mutants was nearly wild-type at eclosion, it declined swiftly with advancing adult age, to be followed by periods of unmoving rigidity and then by premature death. The cellular basis of these phenotypes remains unclear. The neuron-specific pattern of p35 expression, however, and the ability to mimic the lifespan phenotype of p35 null mutants by specific expression of dominant negative Cdk5 in postmitotic neurons demonstrate that it is the function of p35 in mature, postmitotic neurons that is essential for normal lifespan. Experiments are currently in progress to test whether the lifespan and behavioral phenotypes of p35 are a delayed, secondary consequence of developmental miswiring, or whether they reflect a required adult function of this protein (Connell-Crowley, 2007).
It is noteworthy that one of the obvious behavioral defects in p35 mutant adults is a tendency of the adult flies to lie on their backs, unable to right themselves. This phenomenon has been observed previously in the Mediteranean fruit fly, Ceratitis capitata, and has been termed the 'supine' phenotype. In that system, as well, the behavior was associated with reduced lifespan and it was further shown that assumption of the supine phenotype by a particular individual was an early marker of approaching death (Connell-Crowley, 2007).
The pattern of behavioral phenotypes observed in p35 null adults bear intriguing parallels with observable features of mammalian neurodegenerative diseases, some of which have been linked to altered activity of mammalian Cdk5. Does neurodegeneration play a role in the progressive phenotype of p35 null mutants? Can the wild-type functions and mutant phenotypes of p35 and Cdk5 in Drosophila shed any light on mammalian neurodegenerative diseases? If detailed histological and ultrastructural analysis of p35 mutants reveals anatomical degeneration with morphological similarity to mammalian neurodegeneration, it holds the promise of applying the uniquely precise tools of Drosophila genetics to investigate fundamental cellular and molecular mysteries of neurodegeneration, including the earliest events in neuropathology, cell autonomy, and the molecular genetic basis of the disease process (Connell-Crowley, 2007).
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