Block in Nuclear Localization of Period Protein by a Second Clock Mutation, Timeless LB Vosshall, JL Price, a Sehgal, L Saez and MW Young

:5;.2f·f.)%5;%S.C.//i:.;'.2;.·..:.;.:..

bash, J. C. Hall, Neuron 1,141(1988); D. M. Zerr, edge L. Vosshall, L. Saez, C. Wesley, M. Myers, cal advice on RNase protections. Supported by J. C. Hall, M. Rosbash, K. K. Siwicki, J. Neurosci. T. Lieber, and S. Kidd for fruitful discussions and the Howard Hughes Medical Institute and the 10, 2749 (1990); I. Edery, L J. Zwiebel, M. E. critical reading of the manuscript. Excellent National Science Foundation Science and Tech- Dembinska, M. Rosbash, Proc. Natl. Acad. Sci. technical assistance has been provided by M. nology Center for Biological Timing. U.S.A. 91, 2260 (1994). Markowitz, H. Radziewicz, E. Alcamo, and T. 7. S. T. Crews, J. B. Thomas, C. S. Goodman, Cell Evering. M. Baylies graciously provided techni- 12 November 1993; accepted 27 January 1994 52, 143 (1988); E. C. Hoffman et al., Science 252, 954 (1991); K. M. Burbach, A. Poland, C. A. Bradfield, Proc. Natl. Acad. Sci. U.S.A. 89, 8185 (1992); H. Reyes, S. Reisz-Porszasz, O. Hankin- son, Science 256, 1193 (1992). Block in Nuclear Localization of period Protein 8. Z. J. Huang et al., Nature 364, 259 (1993). 9. P. E. Hardin, J. C. Hall, M. Rosbash, ibid. 343, 536 by a Second Clock Mutation, timeless (1990); Proc. Natl. Acad. Sci. U.S.A. 89, 11711 (1992). 10. L. Cooley et al., Science 239, 1121 (1988). Leslie B. Vosshall,* Jeffrey L. Price, Amita Sehgal,t 11. H. M. Robertson et al., Genetics 118, 461 (1988). Lino Michael W. 12. A. C. Spradling and G. M. Rubin, Cell 34, 47 Saez, Youngt (1983). In the is found in nuclei of the and 13. A. Sehgal, J. L. Price, B. Man, M. W. Young, wild-type Drosophila, period protein (PER) eyes brain, unpublished results. and PER immunoreactivity oscillates with a circadian rhythm. The studies described here 14. C. Helfrich and W. Engelmann, Z Naturforsch. 42C, indicate that the nuclear localization of PER is blocked by timeless (tim), a second chro- 1335 (1987); M. J. Hamblen-Coyle et al., J. Insect. mosome mutation that, like null mutations, abolishes circadian PER fusion Behav. 5,417(1992); D. A. Wheeler, M. J. Hamblen- per rhythms. Coyle, M. S. Dushay, J. C. Hall, J. Biol. Rhythms 8, proteins without a conserved domain (PAS) and some flanking sequences are nuclear in 67 (1993). tim mutants. This suggests that a segment of PER inhibits nuclear localization in tim 15. M. Hamblen et al., J. Neurogenet. 3, 249 (1986); mutants. The tim gene may have a role in establishing rhythms of PER abundance and Downloaded from H. B. Dowse, J. C. Hall, J. M. Ringo, Behav. in Genet. 17, 19 (1987). nuclear localization wild-type flies. 16. Hybrid dysgenesis can induce P element excision mutations that ultimately have few or no P element sequences. See M. G. Kidwell, in Drosophila: A PracticalApproach, D. B. Roberts, Ed. (IRL Press, Oxford, 1986), chap. 3; S. B. Daniels and A. Mutations in the Drosophila period (per) sponding changes in the rhythms of per

Chovnick, Genetics 133, 623 (1993); and W. R. gene disrupt circadian rhythms of pupal RNA and protein amounts (10-12) and http://science.sciencemag.org/ Engels, D. M. Johnson-Schlitz, W. B. Eggleston, J. eclosion and adult locomotor behavior PER in nuclei This Sved, Cell62, 515 (1990). (1). immunoreactivity (8). 17. Wild-type (Canton S), per', and tim flies were Although per has been cloned and se- suggests a possible role for molecular oscil- contemporaneously entrained as follows: 0- to quenced and its pattern of expression has lations ofper in the establishment ofbehav- 5-day-old adult flies were transferred to fresh been the biochemical func- ioral a new muta- bottles and maintained in LD 12:12 at 25°C for 3 analyzed (2, 3), rhythms (11). Recently, days. Beginning on the fourth day, collections tion of the PER protein is unknown. PER tion, timeless (tim), was isolated that pro- were made at ZT2, 6, 10, 14, 18, and 22 (ZTO = shares some homology with a family of duces arrhythmic behavior and suppresses lights on). Each collection consisted of shaking transcription factors (4-6) that possess a the circadian oscillation of per RNA (12). approximately 1500 flies into a 15-ml conical tim polypropylene tube kept in dry ice. Total RNAwas common sequence motif called the PAS Here, we examine the effect of on the extracted from the heads, which we separated domain. The PAS domain consists of two expression and localization of PER protein. from the bodies by shaking frozen flies in sieves at of 50 amino acids We PER in repeats approximately compared protein expression temperatures below -20°C. A 32P-labeled ribo- within a of 258 to 308 and tim mutant flies on October 12, 2017 probe that protects 316 nucleotides (nt) of per homology region wild-type, per0 mutant, mRNA (nt 123 to 438 of the per mRNA coding amino acids (7). by staining head sections with PER anti- sequence) (3) was transcribed from cloned per Immunocytochemical experiments dem- body. Because the amounts of PER protein DNA. A tubulin riboprobe also was produced that onstrated that PER is a nuclear in a in and brain nuclei fluctuate protects 86 nt (nt 57 to 142 from the start of protein staining eye transcription) of al tubulin mRNA (19). Ribonu- variety of Drosophila tissues (8, 9). In cells daily (8), sections were prepared at four clease (RNase) protection experiments were car- of the adult fly visual and nervous systems, time points. Nuclear staining in wild-type ried out as described (20) with minor variations. the amount of PER fluctuates with a cells was most at Ten micrograms of head RNA was used for each protein photoreceptor prominent time point, and digestions with RNase were for 30 circadian rhythm (10), the protein is phos- Zeitgeber times 2 and 20 (ZT2 and 20) min at 25°C. Hybridizations and RNase digestions phorylated with a circadian rhythm (10), (13), intermediate at ZT7, and absent at were performed contemporaneously for wild-type, and PER is observed in nuclei at but ZT13 1A). Because the per0 mutation per0, and tim RNA samples representing the first night (Fig. 4 days. For days 5 and 6 of the tim record, RNA not late in the day (8). The expression of introduces a stop codon in the PER reading hybridizations and digestions involved newly la- per RNA is also cyclic. However, peak frame (2, 3), PER antibody specificity was beled riboprobes, so RNA from day 4 was again mRNA amounts are present late in the day, demonstrated by the absence of staining in processed to allow direct comparison of per RNA amounts measured in the two sets of experiments. and the smallest amounts are present late at sections from this null mutant at each time Digested samples were separated by electropho- night (11, 12). Three mutant alleles-per°, point (Fig. 1C). In tim mutants, nuclear resis on 5% polyacrylamide-8 M urea gels. Dried pers, and perL-cause arrhythmic behavior staining was not seen at any time point gels were exposed to x-ray film and also analyzed or shorten or with a phosphorimager (Molecular Dynamics) for lengthen periods, respectively (Fig. 1B). quantitation. (1). These mutations also produce corre- PER-P-galactosidase (PER-P3-gal) fusion 18. L B. Vosshall, J. L Price, A. Sehgal, L. Saez, M. proteins have been used extensively to study W. Young, Science 263, 1606 (1994). of per locus 19. W. E. Theurkauf, H. Baum, J. Bo, P. C. Wensink, Howard Hughes Medical Institute, National Science patterns expression (9, 14, 15). Proc. Natl. Acad. Sci. U.S.A. 83, 8477 (1986). Foundation Science and Technology Center for Bio- Such fusion proteins have also allowed the 20. K. Zinn, D. DiMaio, T. Maniatis, Cell 34, 865 logical Timing, and Laboratory of Genetics, The Rock- functional dissection of the gene and the (1983). efeller University, New York, NY 10021, USA. encoded protein (9, 14, 15). To explore the 21. A. Sehgal, J. Price, M. W. Young, Proc. Natl. *Present address: Center for Neurobiology and Be- effect of tim on PER and Acad. Sci. U.S.A. 89, 1423 (1992). havior and Howard Hughes Medical Institute, Colum- protein expression 22. We gratefully acknowledge the contributions of bia University College of Physicians and Surgeons, to map elements of the per locus responding many single P element insertion lines obtained New York, NY 10032, USA. to tim, we compared patterns ofexpression of from the laboratories of L. Y. Jan and Y. N. Jan fPresent address: Department of Neuroscience, Uni- a fusion in and C. Montell, J. Carlson, and A. Spradling, as versity of Pennsylvania School of Medicine, Philadel- PER-P-gal protein (PER-SG) (9) well as the contribution of a Drosophila tubulin phia, PA 19104, USA. transgenic flies with a wild-type or tim mu- probe from V. Corbin. We would like to acknowl- :To whom correspondence should be addressed. tant genetic background. PER-SG contains 1606 SCIENCE * VOL. 263 · 18 MARCH 1994 .L.P.P.I.R.91I.I.PBIPI the NH2-terminal half of PER (amino acids and tim mutant flies, we prepared protein protein was predicted for both genetic back- 1 to 636) (2) fused to P-gal. The expression immunoblots from tim+, PER-SG and tim, grounds (16-18). PER-SGA96-529 was nu- of PER-SG in transgenic flies in vivo (9) PER-SG head extracts. PER-3-gal fusion clear in wild-type and tim flies (17). The closely matches that of the endogenous pro- proteins of the same apparent size and localization of PER1-95p-gal also was nu- tein (8). Whereas PER-SG localized to nu- abundance were detected in the two genetic clear in wild-type (Fig. 3A, left) and tim clei in wild-type flies, we found that nuclear backgrounds (Fig. 2G). Thus, effects on backgrounds (Fig. 3A, right), which indi- localization was blocked in tim mutants. nuclear localization observed immunocy- cates that sequences in this NH2-terminal Although no nuclear staining was observed tochemically in tim flies are not the result of region ofPER would promote nuclear local- in tim mutants, PER-SG accumulated in the reduced expression or increased proteolysis ization of a full-length PER protein but are same tissues in both tim and wild-type flies of the fusion protein. unable to do so in tim mutant flies unless (Fig. 2, A and D, and Fig. 3B). Specificity of To explore the specificity of the block in another, more centrally located region of the P-gal antibody used in these studies was nuclear localization in tim mutants, we PER is deleted. PER1-636ANLS[66-79]lS- indicated by the complete absence of stain- examined the expression in tim mutants of gal localized to nuclei in wild-type tissue ing in sections derived from wild-type or tim two P3-gal marker proteins (containing no (Fig. 3C, left), which indicates that PER flies lacking the transgene that encodes PER- PER sequences) that had been modified by contains additional nuclear localization sig- SG (16). the addition of heterologous nuclear local- nals. PER1-636ANLS[66-79]pf-gal failed to To determine whether a single mutation ization signals and are expressed in the enter nuclei in a tim background (Fig. 3C, causes both aberrant intracellular localiza- nuclei ofthe eye and brain. The addition of right). Together, these results suggest that tion of PER and the arrhythmic behavior of such nuclear localization signals is required wild-type tim activity is required for nuclear tim flies, we generated recombinants be- for nuclear localization of P-gal in wild-type localization of proteins that contain a spe- tween the second chromosome bearing the flies (16-18). Nuclear localization of these cific region of PER. Because both PER1- Downloaded from PER-SG construct and the tim mutant marker proteins was unaffected by tim (Fig. 95p-gal and PER-SGA96-529 are nuclear chromosome. Head sections prepared from 3, D and E). The tim mutation affected in tim flies, the tim-sensitive region of PER each of 36 homozygous tim+ or tim recom- neither the morphology nor the number of must be located between amino acids 96 binant lines (17 tim+ and 19 tim) were nuclei visualized by these markers. and 529. This interval includes the site of stained with 3-gal antibody. The PER-SG We also examined the localization of the per1- mutation and the PAS domain (2, fusion protein was nuclear in all 17 tim+, four additional PER-P-gal fusion proteins 3), but would not include the per' domain, http://science.sciencemag.org/ PER-SG recombinant lines. Nuclear local- in tim mutants and wild-type flies. Fusion a region in which mutations predominantly ization was not observed in any ofthe 19 tim, proteins contained the first 23 amino acids confer short-period phenotypes (19). PER-SG recombinants (16, 17). Therefore, of PER (PER1-231p-gal), PER amino acids We have drawn four principal conclu- mutant behavioral and localization pheno- 1 to 95 and 530 to 636 (PER-SGA96-529), sions from this work. (i) The tim gene is types are likely to be the result of the same the first 95 amnino acids of PER (PER1-951- required for nuclear localization ofPER. (ii) mutation. Representative sections from gal), or th~ NH2-terminal half of PER Aberrant behavioral and localization phe- three independent tim+, PER-SG recombi- without a putative nuclear localization sig- notypes appear to be a result of the same nants (with nuclear labeling in the photore- nal (PER1-636ANLS[66-79]p-gal) (2). mutation. (iii) Regulation of nuclear local- ceptor, cone, and pigment cells in the eye The pattern ofPER1-23p-gal expression ization by tim may be specific for PER. (iv)

and central in and tim PER contains that somehow in- and in the lamina, medulla, wild-type genetic backgrounds sequences on October 12, 2017 brain) are shown in Fig. 2, A through C. was identical to that of PER-SG in tim hibit PER nuclear localization in the ab- Representative sections from tim, PER-SG mutants (16, 17). Because no sequences sence of tim. A protein encoded by tim recombinants show that nuclear localization resembling a nuclear localization signal are might interact with PER to facilitate nucle- of the PER-SG protein was not observed in found in this segment of the PER protein, ar entry, or tim may regulate factors respon- any of these cells (Fig. 2, D through F). cytoplasmic accumulation of this fusion sible for cytoplasmic retention of PER pro- We examined the intracellular localization of the PER-SG fusion protein in tim/ tim+ heterozygotes to learn whether the ZT2 Z17 ZT13 ZT20 aberrant localization phenotype is a domi- nant or a recessive trait. PER-SG fusion proteins localize to nuclei in the heterozygotes (17). Therefore, like the behavioral defect (12), the nuclear localization defect of tim is recessive. The nuclear localization of PER-SG in tim' flies also is not depen- dent on the presence of endogenous PER protein because its localization was nuclear in a per0 genetic background (17). Thus, neither functional PER protein nor behavioral rhythmicity is required to generate correct nuclear localization of the PER-SG fusion protein in tim+ flies. As well, the tim was inde- Fig. 1. Expression of PER protein in (A) wild-type, (B) tim, and (C) per0 mutant backgrounds at four mutant localization phenotype time points. The nuclear layer is marked with open arrows. Structures underlying the lens frequently pendent of endogenous PER, because PER- stain nonspecifically and do not reflect PER antibody staining in nuclei (black arrowheads). Flies SG fusion protein did not localize to nuclei were raised at 25°C in a 12-hour light:12-hour dark cycle (LD 12:12). Frozen sections (14 p.m) from in per0; tim double mutants (17). adult fly heads collected at the indicated times were incubated with PER antibody (1:30 dilution of To search for possible differences in the immune serum with PER antibody 19-06 raised against full-length PER protein and preabsorbed rate of synthesis, stability, or structure of against per0 head lysate) for 16 hours at room temperature. Antibody staining was continued as PER-SG proteins isolated from wild-type described (8) with the use of the Vectastain ABC kit (Vector Labs). SCIENCE * VOL. 263 * 18 MARCH 1994 1607 Bl.llllllilsll.s.lDI... tein. The arrhythmic behavior and lack of cycling of PER nuclear localization. formation of a complex with the ARNT per RNA rhythms observed in tim mutants The region implicated in tim control of subunit and promotes nuclear localization of (12) are likely to be a direct consequence nuclear localization of PER includes the the receptorcomplex (22). In the absence of of the failure of PER to enter the nucleus. PAS domain, nonconserved sequences up- either ligand or ARNT, the receptor fails to Thus, PER must be present in the nucleus stream of PAS (amino acids 96 to 232) enter nuclei and has no transcriptional ac- at some time of day for expression of (20), and 33 amino acids downstream of tivity (5, 23). No ligands or binding partners circadian rhythms. Our detection of large PAS (amino acids 497 to 529) (4, 14). amounts of cytoplasmic PER-3-gal fusion Regulation of nuclear localization has been protein, but not full-length cytoplasmic observed for two other proteins containing WHd type #rmeles PER protein, in tim mutants may also the PAS domain. The dioxin receptor is a indicate that PER is unstable in the cyto- ligand-activated transcription factor (21) plasm and stabilized by transport to the that accumulates in the cytoplasm until nucleus. Thus, the observed cycling of dioxin ligand interacts with the PAS domain PER protein abundance (10) might reflect of the AHR subunit (6). This induces the Downloaded from http://science.sciencemag.org/

on October 12, 2017 Fig. 2. Immunocytochemistry and protein im- G munoblot analysis of a PER-3-gal fusion pro- " c tein (PER-SG) (9) in tim+ and tim recombinant lines. Shown is expression of PER-SG in head - ^ . X. x x Fig. 3. Localization of PER-13-gal and 13-gal sections from tim+ (A through C) and tim mu- marker proteins in wild-type (left column) and tant (D throughFl recombinant lines. For (A) tim mutant (right column) adult head sections. through (C), staining of nuclei was observed in (A) PER1-9513-gal. (B) PER1-63613-gal (identi- photoreceptor cells (ret), lamina (lam), medulla 200- cal in amino acid composition to PER-SG, but (med), and some cells of the central brain. constructed as described below). (C) PER1- Open arrows indicate nuclear labeling. In (D) 636ANLS[66-79113-gal. (D) Enhancer trap line through (F), staining of photoreceptor cells and S45 (expresses -gal modified by the addition cells of the lamina, medulla, and central brain of a nuclear localization signal in the eye and a was also observed, but there is no indication of few cells in the brain). (E) Eye protein kinase nuclear localization of the stain. Frozen sec- 07.4- C-P-gal (1-gal modified by the addition of the tions were incubated with affinity-purified E3-gal SV40 nuclear localization signal under the con- polyclonal antibody (Cappel/Organon Tech- trol of the eye-specific protein kinase C promot- nika, Durham, North Carolina) (1:1000 dilution er) (18). Staining with antibodies to 13-gal was preabsorbed against adult fly acetone powder) as described in legend to Fig. 2. PER-p-gal and processed as described (Fig. 1). (G) Lines fusion constructs were made by inserting an of tim and+tim mutant flies containing PER-SG Xho I linker at nucleotide 3183 (PER1-9513-gal; or a control line (Canton-S) lacking the trans- Ace I site) or at nucleotide 4936 (PER1-63613- gene, were raised as described (Fig. 1). Fies (6 to 12 days old) were collected from ZT18 to ZT22. gal and PER1-636ANLS[66-79]13-gal; Sac I Extracts were prepared from the heads of flies as described (15). Extract containing 1 x (10 I.g by site) (2). PER1-636ANLS[66-79]p-gal is iden- Bradford assay), or the indicated fraction of total protein, was electrophoresed in each lane of an tical to PER1-63613-gal except that a fragment SDS-polyacrylamide gel (24) and analyzed for 13-gal antigen levels as described by Saez and encoding amino acids 65 to 95 has been de- Young (8). The primary antibody was a mouse 13-gal monoclonal antibody, and the secondary leted in the former (2, 16). The Sph I-Xba I per antibody was goat antibody to mouse immunoglobulin G (heavy and light chains) alkaline promoter fragment was added and the per phosphatase conjugate (both from Promega). Extracts from tim, PER-SG flies were loaded in the fragment excised with Xho I. All constructs first four lanes; tim+, PER-SG extract in the fifth lane; and extract from a line that does not contain were cloned into pCaSpeR-13-gal (25), altered the PER-SG transgene (Canton-S) in the sixth lane. Molecular size markers (Amersham) are to create three different frames with Xho I link- indicated to the left of the gel. ers (16). 1608 SCIENCE * VOL. 263 * 18 MARCH 1994 i.i.i.-.-:.:.:...:.S.:.:...:...:.:.:.:.i"":.. :·.s:.:.:.:.:.:.:...:...:.r.S:.:...:.:.j:.i.:.:.:.`.:.:.::a8#:..:.:.:.:.:..'' ""'". :·t·:i·:·.t:·:::.ic.·:.:::.::·:·:·:.:::...r...·; .i.s.iS.i.iiii.i.i.::::::5.:.:I::.:.::·····:·.i.:.:.:.:#s.:'ri·i.·.·..·rz;z:v;·2:·I.-.-.;-.-.;------·;;-,, ::::::.:r.s...... i.i...:...:.:..::·..:.:.:.:.:.:.:.:....r··i·i.i.Y '"''· ''"·.;.·.·.·...·.·..·.·.·.·...'"'" ''""""""""".5.:.....·....i.i.i.iiiii.F.:.:.:.:.:.j.:..i.i.:.::I.·z.- iii...i. 'n·..SS.X..:.::.:::.::::.I:o:.i.i.i.l.:.: . ...:.:.::.:·:·.·:····._XS·W:..:...S.:.:.:._:.i·.·:.:.·:·.:·(··2···i···i· ·ii·L·i ······f:·IXX.·:·r·S·;·2····j.....i.:.:.:.:.:.:.:3:·(·:.·..·i·; .iii·s. ..:o·iaa:i:··. "':':S.:.:.:.:.:.i::.i.:.::.:.:i::.:.?., I.auolo to ARNT have been identified for 6. K. M. Burbach, A. Poland, C. A. Bradfield, Proc. Rosbash, Proc. Natl. Acad. Sci. U.S.A. 88, 3882 analogous Natl. Acad. Sci. U.S.A. 89, 8185 (1992). (1991). PER. However, it has recently been shown 7. Sequence similarity between PER and a protein 16. L. B. Vosshall, thesis, Rockefeller University that the PAS domain ofthe PER protein can regulating aspects of Drosophila development, (1993). function as a protein interaction domain SIM (encoded by the gene single-minded), was 17. L. B. Vosshall, J. Price, M. W. Young, unpublished first recognized by Crews et al. (4). Subsequent observations. (14). Further mapping of the tim-sensitive studies have shown that this homology region is 18. D. P. Smith etal., Science 254, 1478 (1991). region of PER should determine whether also found in ARNT (arylhydrocarbon receptor 19. M. K. Baylies, L. B. Vosshall, A. Sehgal, M. W. PAS is centrally involved in the control of nuclear translocator), a component of the multi- Young, Neuron 9, 575 (1992). PER nuclear localization. subunit mammalian dioxin receptor (5), and in 20. H. V. Colot, J. C. Hall, M. Rosbash, EMBO J. 7, AHR, the ligand-binding subunit of the dioxin 3929 (1988). Peak amounts of per RNA expression receptor (6). The region of homology is now 21. M. S. Denison, J. M. Fisher, J. P. Whitlock Jr., (11, 12) precede the greatest amount of referred to as the PAS domain, after the first Proc. Natl. Acad. Sci. U.S.A. 85, 2528 (1988); J. PER in nuclei of the three proteins in which it was identified: PER, Hapgood, S. Cuthill, M. Denis, L. Poellinger, J. A. protein staining pho- ARNT, and SIM (14). Gustafsson, ibid. 86, 60 (1989). toreceptors and brain (8) by about 8 hours. 8. K. K. Siwicki, C. Eastman, G. Petersen, M. Rosbash, 22. A. Fujisawa-Sehara, M. Yamane, Y. Fujii- Thus, nuclear immunoreactivity of PER J. C. Hall, Neuron 1,141 (1988); L. Saez and M. W. Kuriyama, ibid. 85, 5859 (1988); A. B. Okey et al., occurs when the amount of RNA is Young, Mol. Cell. Biol. 8, 5378 (1988); D. M. Zerr, J. J. Biol. Chem. 255, 11415 (1980). per C. Hall, M. Rosbash, K. K. Siwicki, J. Neurosci. 10, 23. S. O. Karenlampi, C. Legraverend, J. M. Gudas, small. As suggested above, the entry of PER 2749 (1990). N. Carramanzana, O. Hankinson, J. Biol. Chem. protein into the nucleus may be under 9. X. Liu, L. Lorenz, Q. Yu, J. C. Hall, M. Rosbash, 263, 10111 (1988). control, so that some of the Genes Dev. 2,228 (1988); X. Liu etal., Neuron 6, 24. U. K. Laemmli, Nature 227, 680 (1970). temporal pre- 753 (1991); X. Liu et al., J. Neurosci. 12, 2735 25. C. S. Thummel, A. M. Boulet, H. D. Lipshitz, Gene viously observed oscillation in PER protein (1992); J. Ewer, B. Frisch, M. J. Hamblen-Coyle, 74, 445 (1988). amounts and nuclear staining (8, 10) may M. Rosbash, J. C. Hall, ibid., p. 3321. 26. Fly stocks were kindly provided by the following reflect rhythmic movement between the 10. I. Edery, L. J. Zwiebel, M. E. Dembinska, M. investigators: J. Treisman, Z. Lai, and G. M. Rubin with a Rosbash, Proc. Natl. Acad. Sci. U.S.A. 91, 2260 (S45 enhancer trap line); D. P. Smith and C. S. Downloaded from cytoplasm and the nucleus, phase (1994). Zuker (eye protein kinase C-NLS-p-gal trans- that is distinct from that of per RNA syn- 11. P. E. Hardin, J. C. Hall, M. Rosbash, Nature 343, formant lines 5201 and 5202); and M. Rosbash thesis. In light of these observations and 536 (1990); Proc. Natl. Acad. Sci. U.S.A. 89, (PER-3-gal transformant line PER-SG). We thank D. our work with we that forma- 11711 (1992). Sulzer, T. Lieber, M. Myers, M. E. Hatten, and R. Axel tim, speculate 12. A. Sehgal, J. L. Price, B. Man, M. W. Young, for critically reading the manuscript. Supported by tion of an intracellular circadian clock may Science 263, 1603 (1994). the Howard Hughes Medical Institute and the Na- require nuclear localization of PER to be 13. Zeitgeber time (ZT) is an arbitrary standard for tional Science Foundation Science and Technology limited to a time of measuring time; ZTO is defined as lights on Center for Biological Timing. The work of L.B.V. was http://science.sciencemag.org/ particular day. or dawn, and ZT12 is defined as lights off or dusk. partially funded by National Research Service In such a model, constitutively cytoplas- 14. Z. J. Huang, I. Edery, M. Rosbash, Nature 364, Awards training grant GM07982-09. mic (as in tim mutants) or constitutively 259 (1993). nuclear PER would fail to generate circa- 15. L J. Zwiebel, P. E. Hardin, X. Liu, J. C. Hall, M. 12 November 1993; accepted 27 January 1994 dian rhythms, and tim' activity might play a role in temporal regulation of the access of PER to the nucleus. It was proposed that Activation of PER may directly or indirectly regulate its Phosphatidylinositol-3' Kinase by own transcription, because the cycling of Src-Family Kinase SH3 Binding to the p85 Subunit per transcription is blocked in per0 mutants

Because the tim mutation also abol- (11). M. W. Marc John C. Cambier* on October 12, 2017 ishes these RNA rhythms (12), a form of Christopher Pleimian, Hertz, feedback regulation may exist in which Engagement of antigen receptor complexes induces rapid activation of Src-family kinases cycling nuclear localization of PER might and association with phosphatidylinositol-3' kinase (PI-3 kinase). Here it was found that produce rhythmic signals influencing per the Src homology 3 (SH3) domain of Lyn and Fyn bound to a proline-rich region (residues transcription. We wish to emphasize that 84 to 99) within the 85-kilodalton subunit (p85) of PI-3 kinase. The binding of SH3 to the tim was recovered in a screen for clock purified kinase led to a five- to sevenfold increase in the specific activity of PI-3 kinase. mutations that was not biased to afford Ligand-induced receptor stimulation activated PI-3 kinase, and this activation was blocked recovery of new mutations interacting with by a peptide containing residues 84 to 99 of p85. These data demonstrate a mechanism per (12). Thus, the discovery of a function- for PI-3 kinase activation and show that binding of SH3 domains to proline-rich target al interaction between tim and per indicates sequences can regulate enzymatic activity. that a single intracellular mechanism is probably central to the generation of circadian rhythms in Drosophila. The effects of tim on per RNA oscillation and PER nucle- The response of lymphocytes to antigen is receptors causes activation of the receptor- ar localization would presumably be compo- mediated through a multisubunit cell sur- associated Src-family tyrosine kinases p55blk nents of this mechanism. face receptor complex. Antigen receptor (Blk), p59hnf (Fyn), and p53/56'y" (Lyn) engagement can initiate multiple intracel- (2). Lyn associates with the noncatalytic REFERENCES AND NOTES lular signaling events that lead to activa- 85-kD subunit of PI-3 kinase in B cells after tion, differentiation, and tolerance induc- (3). 1. R. J. Konopka and S. Benzer, Proc. Natl. Acad. antigen receptor ligation Sci. U.S.A. 68, 2112 (1971). tion, depending on the nature of the stim- The PI-3 kinase is a heterodimeric pro- 2. M. K. Baylies, L. Weiner, L. B. Vosshall, L. Saez, ulus and differentiative stage of the cell (1). tein composed of a noncatalytic p85 sub- M. W. Young, in Molecular Genetics of Biological The binding of ligand to B cell antigen unit (4), catalytic 110-kD subunits (p110) Rhythms, M. W. Young, Ed. (Dekker, New York, and inositol on 1993), pp. 123-153. (5), phosphorylated lipids 3. M. Rosbash and J. C. Hall, Neuron 3, 387 (1989). Division of Basic Sciences, Department of Pediatrics, the D-3 hydroxyl position (6). The exact 4. S. T. Crews, J. B. Thomas, C. S. Goodman, Cell National Jewish Center for Immunology and Respira- roles of the phosphoinositide products gen- 143 J. R. J. K. A. tory Medicine, 1400 Jackson Street, Denver, CO 52, (1988); Nambu, O. Lewis, and erated by PI-3 kinase in signaling pathways Wharton Jr., S. T. Crews, ibid. 67, 1157 (1991). 80206, USA, Department of Microbiology and have not been 5. H. Reyes, S. Reisz-Porszasz, O. Hankinson, Sci- Immunology, University of Colorado Health Science determined, but they accu- ence 256, 1193 (1992); E. C. Hoffman et al., ibid. Center, Denver, CO 80206, USA. mulate in cells activated by growth factors 252, 954 (1991). *To whom correspondence should be addressed. (7). PI-3 kinase interacts with SV40 middle SCIENCE · VOL. 263 * 18 MARCH 1994 1609 Block in nuclear localization of period protein by a second clock mutation, timeless LB Vosshall, JL Price, A Sehgal, L Saez and MW Young

Science 263 (5153), 1606-1609. DOI: 10.1126/science.8128247 Downloaded from ARTICLE TOOLS http://science.sciencemag.org/content/263/5153/1606

REFERENCES This article cites 29 articles, 18 of which you can access for free http://science.sciencemag.org/content/263/5153/1606#BIBL http://science.sciencemag.org/

PERMISSIONS http://www.sciencemag.org/help/reprints-and-permissions

on October 12, 2017

Use of this article is subject to the Terms of Service

Science (print ISSN 0036-8075; online ISSN 1095-9203) is published by the American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. 2017 © The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. The title Science is a registered trademark of AAAS.