Biochemical and phenotypic analysis of the p21-activated kinase DPAK3 in Drosophila melanogaster

Gegenstand dieser Arbeit ist das Drosophila melanogaster Protein DPAK3, ein Vertreter der hochkonservierten Familie der p21-aktivierten Kinasen (PAK). DPAK3 und seine Homologen aus anderen Insektenarten und C. elegans können aufgrund eines Vergleichs der Proteinsequenz und struktureller Merkmale in eine eigenen Untergruppe 1* innerhalb der Gruppe 1 der PAK-Proteine eingeordnet werden. Das Genom von Drosophila kodiert noch für zwei weitere PAK-Proteine, das zur Gruppe 1 gehörende DPAK1 und das Gruppe 2 PAK-Protein Mbt. Wie die klassischen Gruppe 1 PAK-Proteine bildet DPAK3 im inaktiven Zustand Dimere. DPAK3 interagiert mit den GTP-gebundenen Formen der RhoGTPasen Rac1, Rac2 und Cdc42. Durch die Bindung dieser Proteine geht DPAK3 aus dem dimeren in den monomeren Zustand über und seine Kinaseaktivität wird durch diese Bindung gesteigert. DPAK3 ist für die Ausbildung der korrekten Morphologie kultivierter Drosophila Zellen erforderlich und beeinflußt die Regulation des Aktinzytoskeletts. Weiterhin konnte CK2beta, die regulatorische Untereinheit der Casein Kinase 2, als neuer Regulator von p21-aktivierten Kinasen identifiziert werden. Das Genom von Drosophila besitzt drei Transkriptionseinheiten, die für CK2beta', CK2betatestes und fünf verschiedene Isoformen von CK2beta kodieren. Eine vergleichende Analyse zeigt, daß alle CK2beta-Proteine mit DPAK1, DPAK3 und in geringerem Maß auch mit Mbt interagieren und in der Lage sind, die Aktivität der PAK-Proteine in vitro zu hemmen. Die Bindung von CK2beta an DPAK3 wird, wie bei allen anderen Serin- / Threoninkinasen, die bisher als Interaktionspartner von CK2beta identifiziert wurden, über die Kinasedomäne von DPAK3 vermittelt. Die Bildung des aus zwei katalytischen CK2a und zwei CK2beta Untereinheiten bestehenden CK2-Holoenzyms hängt von der Fähigkeit von CK2beta ab, Dimere zu bilden. Es konnte gezeigt werden, daß die Bildung eines b-b Dimers für die Interaktion mit und Regulation von DPAK3 nicht erforderlich ist. In vivo wurden die bisher bekannten Dpak3 Allele untersucht, wobei kein gesichertes Nullallel identifiziert werden konnte. Durch enzymatisch katalysierte Rekombination wurde eine neue Deletion hergestellt, die das komplette Leseraster von Dpak3 entfernt. Mit Hilfe von genetischen Mosaiken wurde die Rolle von DPAK3 in der Augenentwicklung untersucht. Durch den Verlust der Genfunktion von Dpak3 wird die Ausbildung der korrekten Struktur der Komplexaugen nur leicht beeinträchtigt. Bei der Analyse einer Dpak1 Mutante wurde dasselbe Ergebnis erzielt. Gleichzeitiger Verlust der Genfunktion von Dpak1 und Dpak3 hingegen führt zu massiven strukturellen Defekten. DPAK1 und DPAK3 erfüllen somit zumindest teilweise redundante Funktionen in der Augenentwicklung. Es wird Gegenstand zukünftiger Studien sein müssen, die gemeinsamen und getrennten Funktionen dieser PAK-Proteine in Drosophila aufzuklären.Subject of this work is the Drosophila melanogaster protein DPAK3, a member of the highly conserved family of p21-activated kinases. Based on the comparison of the amino acid sequence and structural features, DPAK3 and its homologues from other insect species and C. elegans can be assigned to a distinct subgroup 1* within the group 1 PAK proteins. The genome of Drosophila encodes for two additional PAK proteins, DPAK1 and Mbt, which belong to the group 1 and group 2 p21-activated kinases, respectively. Like the classical group 1 PAK proteins, DPAK3 forms dimers in its inactive conformation. DPAK3 binds to and is activated by the Rho GTPases Rac1, Rac2 and Cdc42. The interaction with these proteins leads to the disruption of the DPAK3 dimer and an increase in the kinase activity of DPAK3. DPAK3 is necessary for the development of the normal morphology of cultured Drosophila cells and influences the regulation of the actin cytoskeleton. CK2b the regulatory subunit of casein kinase 2 was identified as a new regulator of p21 activated kinases. The genome of Drosophila possesses three different transcriptional units that encode the proteins CK2b', CK2btestes and five different isoforms of CK2b. A comparative analysis shows that all CK2b proteins interact with DPAK1, DPAK3 and Mbt and negatively regulate the activity of these kinases in vitro. CK2b binds to the kinase domain of DPAK3 which is consistent with previous results obtained from other serine/threonine kinases interacting with CK2b. The CK2 holoenzyme consists of two catalytically active CK2a subunits and two regulatory CK2b subunits. My results show that the ability of CK2b to form dimers, which is essential for the formation of the CK2 holoenzyme, is not necessary for the regulation of p21 activated kinases. The analysis of the available Dpak3 alleles in vivo revealed the necessity to create a new bona fide loss of function allele. To accomplish this goal, a new deletion which removes the entire Dpak3 open reading frame was created by enzymatically catalysed recombination. Genetic mosaics were used to study the role of DPAK3 in eye development. The morphology of the complex eyes was only slightly impaired by the loss of Dpak3 function. The same result was obtained when analysing Dpak1 mutants, but removal of the gene function of both Dpak1 and Dpak3 leads to massive structural defects. This shows that Dpak1 and Dpak3 have at least partially redundant functions in eye development. Further studies will be necessary to reveal the common and distinct functions of these p21-activated kinases in Drosophila

The MAP Kinase p38 Is Part of Drosophila melanogaster's Circadian Clock

All organisms have to adapt to acute as well as to regularly occurring changes in the environment. To deal with these major challenges organisms evolved two fundamental mechanisms: the p38 mitogen-activated protein kinase (MAPK) pathway, a major stress pathway for signaling stressful events, and circadian clocks to prepare for the daily environmental changes. Both systems respond sensitively to light. Recent studies in vertebrates and fungi indicate that p38 is involved in light-signaling to the circadian clock providing an interesting link between stress-induced and regularly rhythmic adaptations of animals to the environment, but the molecular and cellular mechanisms remained largely unknown. Here, we demonstrate by immunocytochemical means that p38 is expressed in Drosophila melanogaster's clock neurons and that it is activated in a clock-dependent manner. Surprisingly, we found that p38 is most active under darkness and, besides its circadian activation, additionally gets inactivated by light. Moreover, locomotor activity recordings revealed that p38 is essential for a wild-type timing of evening activity and for maintaining ∼ 24 h behavioral rhythms under constant darkness: flies with reduced p38 activity in clock neurons, delayed evening activity and lengthened the period of their free-running rhythms. Furthermore, nuclear translocation of the clock protein Period was significantly delayed on the expression of a dominant-negative form of p38b in Drosophila's most important clock neurons. Western Blots revealed that p38 affects the phosphorylation degree of Period, what is likely the reason for its effects on nuclear entry of Period. In vitro kinase assays confirmed our Western Blot results and point to p38 as a potential "clock kinase" phosphorylating Period. Taken together, our findings indicate that the p38 MAP Kinase is an integral component of the core circadian clock of Drosophila in addition to playing a role in stress-input pathways

Daily p38 mRNA (A) and protein expression (B–D) in <i>Canton S</i> wildtype.

<p>A: Quantitative real-time PCR on head extracts revealed constant mRNA expression throughout the day with allover higher levels of <i>p38b</i> compared to <i>p38a</i> (p<0.001). B: Antibody staining with anti-p-p38 on adult brains displayed rhythmic phosphorylation of p38 in DN_1as in LD with significant higher p-p38 levels occurring during the night than in the day (p<0.05). C: A highly significant reduction of active p38 in DN_1as at CT6 compared to CT18 in DD indicates a clock-controlled activation of p38 (p<0.001) D: Only a 15 minute light pulse (LP) during subjective night (CT18) and not during the subjective day (CT6) leads to a reduction in active p38 in DN_1as, suggesting a clock-dependent photic reduction of active p38. The “C” in D indicates control brains without 15 minute light pulse (LP). Error bars show SEM. Significant differences (p<0.05) are indicated by *, highly significant differences (p<0.001) by **.</p

p38b promotes PER phosphorylation during the dark phase.

<p>To analyze daily phosphorylation of PER in flies that express the dominant-negative form of p38b in clock neurons and photoreceptor cells, we performed Western blots on head extracts after 4 days entrainment to LD 12∶12 cycles. According to our behavioral data, timing of PER accumulation was not affected in experimental flies (<i>UAS-p38b^DN-S;cry-Gal4/+;+</i>) in comparison with their respective control (A). However, regarding the degree of PER phosphorylation we observed differences at all time points when we compared both genotypes. For better comparison Western blots were repeated and samples of control and <i>UAS-p38b^DN-S;cry-Gal4/+;+</i> flies were plotted side by side for each ZT (B). Interestingly, flies with impaired p38 signaling indeed had less phosphorylated PER, showing the largest differences to the controls at the end of the night. Western blots were repeated 4 times and always gave similar results. Bars above the blots depict the light regime of the LD 12∶12. The “<i>C</i>” refers to respective control, <i>DNS</i> to <i>UAS-p38b^DN-S;cry-Gal4/+;+</i>.</p

p38b phosphorylates PER <i>in vitro</i>.

<p>To test whether p38b phosphorylates PER <i>in vitro</i>, either non-radioactive kinase assays followed by urea-PAGE (A–D) or radioactive kinase assays with autoradiography (E–F) were performed. A–D: Non-radioactive kinase assays were conducted with poly-histidine tagged p38b (His₆-p38b) and two truncated GST-tagged PER isoforms, GST-PER^1–700 (A,B) and GST-PER^658–1218 (C,D). Samples were subsequently separated with urea-PAGE and visualized by Coomassie staining (A,C). To further confirm PER's position in the gel two samples of the same gel were additionally blotted onto nitrocellulose membrane and immunolabeled using an anti-PER antibody and a secondary fluorescent antibody (B,D). While GST-PER^1–700 without kinase did not shift within 60 minutes, the addition of His₆-p38b induced a downward shift of GST-PER^1–700 indicating phosphorylation of PER (A; dotted line). Immunoblots with anti-PER further confirmed the size as well as the shift of the GST-PER^1–700 band (B). In addition to GST-PER^1–700, GST-PER^658–1218 also displayed band shifts after incubation with His₆-p38b (C). This was most prominent after 60 minutes, when addition of His₆-p38b resulted in two distinct shifted bands (black arrowheads), which could be additionally confirmed by Western blots (D). Time scale below graphs represents minutes after addition of His₆-p38b, the “C” refers to control and represents substrate samples without kinase and ATP. (E) Radioactive <i>in vitro</i> kinase assays were conducted with the indicated GST-PER fusion proteins and GST-p38b. Control reactions were performed in the absence of GST-p38b or with GST in combination with GST-p38b. Coomassie staining proved loading of the indicated protein combinations. Below, phosphorylation of GST-PER proteins was detected by autoradiography. (F) For quantitative analysis five independent <i>in vitro</i> kinase assay experiments were performed and analyzed. For each reaction within a single experiment, autoradiography signal intensities were normalized to the corresponding Coomassie stained protein band. Values in the graph are shown as percentages of GST-PER^658–1218 phosphorylation (100%; * p<0.05, ** p<0.005).</p

Daily oscillations of nuclear PER in s-LN_vs and l-LN_vs of flies expressing a dominant negative form of p38b in these cells.

<p>Flies were entrained in LD 12∶12, dissected every one to two hours and staining intensity of nucleus and cell body was measured as described in Material and Methods. Nuclear PER staining intensities were normalized to total staining and tested for statistically significance. Expression of the dominant negative form of p38b phase delayed nuclear accumulation of PER in the s-LN_vs (A) and l-LN_vs (B). Arrows indicate the maxima of nuclear PER staining that occurred significantly later in <i>UAS-p38b^DN-S;Pdf-Gal4/+;+</i> flies than in control flies. This delay in nuclear PER accumulation in PDF-positive clock neurons is well consistent with the shifted evening activity in these flies. Bars above the graphs depict the light regime of the LD 12∶12.</p

Rhythmicity and period length of all investigated genotypes in constant darkness (DD) according to χ²-periodogram analysis.

<p><i>n</i> indicates the number of tested flies per genotype that survived locomotor recordings. Power and period values were averaged over all rhythmic flies for each genotype.</p>1<p>Flies with power values <20 were defined as arrhythmic.</p>2<p>Power is a measure of rhythmicity and is given in % of variance.</p