A Point Mutation in Nucleoside Diphosphate Kinase Results in a Deficient Light Response for Perithecial Polarity in Neurospora crassa

Abstract

In Neurospora crassa, the phosphorylation of nucleoside diphosphate kinase (NDK)-1 is rapidly enhanced after blue light irradiation. We have investigated the function of NDK-1 in the blue light signal transduction pathway. A mutant called psp (phosphorylation ofsmall protein) shows undetectable phosphorylation of NDK-1 and is defective in light-responsive regulation of perithecial polarity. Sequencing analysis ofndk-1 cDNA by reverse transcription-polymerase chain reaction revealed that proline 72 of ndk-1 was replaced with histidine in psp. The mutation ndk-1P72H resulted in accumulation of normal levels of mRNA and of about 25‥ of NDK-1P72Hprotein compared with that of wild type as determined by Western blot analysis. The ectopic expression of cDNA and introduction of genomic DNA of wild type ndk-1 in psp(ndk-1P72H) suppressed the reduction in accumulation and phosphorylation of NDK-1 and the light-insensitive phenotype. These findings demonstrated that the phenotype ofpsp was caused by the ndk-1P72Hmutation. Biochemical analysis using recombinant NDK-1 and NDK-1P72H indicated that the P72H substitution in NDK-1 was responsible for the decrease in phosphotransfer activities, 5‥ of autophosphorylation activity, and 2‥ of V maxfor protein kinase activity phosphorylating myelin basic protein, compared with those of wild type NDK-1, respectively. In Neurospora crassa, the phosphorylation of nucleoside diphosphate kinase (NDK)-1 is rapidly enhanced after blue light irradiation. We have investigated the function of NDK-1 in the blue light signal transduction pathway. A mutant called psp (phosphorylation ofsmall protein) shows undetectable phosphorylation of NDK-1 and is defective in light-responsive regulation of perithecial polarity. Sequencing analysis ofndk-1 cDNA by reverse transcription-polymerase chain reaction revealed that proline 72 of ndk-1 was replaced with histidine in psp. The mutation ndk-1P72H resulted in accumulation of normal levels of mRNA and of about 25‥ of NDK-1P72Hprotein compared with that of wild type as determined by Western blot analysis. The ectopic expression of cDNA and introduction of genomic DNA of wild type ndk-1 in psp(ndk-1P72H) suppressed the reduction in accumulation and phosphorylation of NDK-1 and the light-insensitive phenotype. These findings demonstrated that the phenotype ofpsp was caused by the ndk-1P72Hmutation. Biochemical analysis using recombinant NDK-1 and NDK-1P72H indicated that the P72H substitution in NDK-1 was responsible for the decrease in phosphotransfer activities, 5‥ of autophosphorylation activity, and 2‥ of V maxfor protein kinase activity phosphorylating myelin basic protein, compared with those of wild type NDK-1, respectively. white collar nucleoside diphosphate kinase reverse transcription polymerase chain reaction 1,4-piperazinediethanesulfonic acid polyacrylamide gel electrophoresis base pair(s) glutathioneS-transferase myelin basic protein Development and morphogenesis during the life cycles of fungi, as well as plants, are greatly affected by environmental stimuli such as light (1Horwitz, B. A., Berrocal, T. G. M., Bot. Acta, 110, 1995, 360, 368.Google Scholar, 2Meyerowitz, E. M., and Somerville, C. R., eds. (1994) Arabidopsis, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar, 3Horwitz, B. A., and Berrocal, T. G. M. (1995) Bot. Acta 110, 360, 368Google Scholar). Recently, the nature of the photoreceptors in fungi and plants has been investigated. In Arabidopsis thaliana, the photoreceptors (4Whitelam G.C. Devlin P.F. Plant Physiol. Biochem. 1998; 36: 125-133Crossref Scopus (31) Google Scholar, 5Batschauer A. Planta. 1998; 206: 479-492Crossref PubMed Scopus (89) Google Scholar) are phytochromes (6Quail P.H. BioEssays. 1997; 19: 571-579Crossref PubMed Scopus (71) Google Scholar), cryptochromes (7Briggs W.R. Huala E. Annu. Rev. Cell Dev. Biol. 1999; 15: 33-62Crossref PubMed Scopus (332) Google Scholar, 8Ahmad M. Jarillo J.A. Smirnova O. Cashmore A.R. Mol. Cell. 1998; 1: 939-948Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar), phototropin (7Briggs W.R. Huala E. Annu. Rev. Cell Dev. Biol. 1999; 15: 33-62Crossref PubMed Scopus (332) Google Scholar, 9Christie J.M. Reymond P. Powell G.K. Bernasconi P. Raibekas A.A. Liscum E. Briggs W.R. 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Rev. 1982; 46: 426-570Crossref PubMed Google Scholar,20Nelson M.A. Morelli G. Carattoli A. Romano N. Macino G. Mol. Cell. Biol. 1989; 9: 1271-1276Crossref PubMed Scopus (107) Google Scholar). Isolation and characterization of wc-1 andwc-2 genes revealed that both of them have a zinc finger DNA-binding motif, a PAS PERIOD, ARNT, and SIM dimerization domain, and a glutamine-rich transcriptional activation domain (21Ballario P. Vittorioso P. Margrelli A. Talora C. Cabibbo A. Macino G. EMBO J. 1996; 15: 1650-1657Crossref PubMed Scopus (375) Google Scholar, 22Linden H. Macino G. EMBO J. 1997; 16: 98-109Crossref PubMed Scopus (286) Google Scholar). WC proteins are proposed to form heterodimers through their PAS domain (23Ballario P. Talora C. Galli D. Linden H. Macino G. Mol. Microbiol. 1998; 29: 719-729Crossref PubMed Scopus (150) Google Scholar). Furthermore, WC-1 has the LOV (light, oxygen, or voltage) domain found in blue light receptor phototropin (7Briggs W.R. Huala E. Annu. Rev. Cell Dev. Biol. 1999; 15: 33-62Crossref PubMed Scopus (332) Google Scholar). These results suggest that WC proteins perceive blue light and regulate the transcription of the blue light-responsive genes. However, it is difficult to explain ion mobilization by WC proteins (12Belozerskaya T.A. Potapova T.V. Exp. Mycol. 1993; 17: 157-169Crossref Scopus (15) Google Scholar). Furthermore, phosphorylation of WC-1 is increased after blue light irradiation (24Talora C. Franchi L. Linden H. Ballario P. Macino G. EMBO J. 1999; 18: 4961-4968Crossref PubMed Scopus (197) Google Scholar), which appears to be an important part of this pathway. However, except for protein kinase C, no candidate for a protein kinase mediating theNeurospora blue light signaling has been suggested (25Arpaia G. Cerri F. Baima S. Macino G. Mol. Gen. Genet. 1999; 262: 314-322Crossref PubMed Scopus (58) Google Scholar). We developed a system to analyze the light-responsive phosphorylation of proteins in vitro utilizing the mycelial membrane fraction. The phosphorylation of a 15-kDa protein increased specifically after blue light irradiation in the wild type, but not in either wc-1 or wc-2. However, mixing the membrane fractions from wc-1 and wc-2 restored the increase in the phosphorylation. These results suggested that the 15-kDa protein was involved in blue light signal transduction downstream of WC proteins (26Oda K. Hasunuma K. FEBS Lett. 1994; 345: 162-166Crossref PubMed Scopus (28) Google Scholar). This protein was purified and identified as nucleoside diphosphate kinase (NDK) (27Ogura Y. Yoshida Y. Ichimura K. Aoyagi C. Yabe N. Hasunuma K. Eur. J. Biochem. 1999; 266: 709-714Crossref PubMed Scopus (21) Google Scholar). The characteristics of an N. crassa mutant known aspsp suggested that it is a mutant allele ofndk-1. Phosphorylation of NDK-1 is undetectable inpsp (see Fig. 1 A), and the psp mutant is known to be defective in light-responsive regulation of perithecial polarity (17Oda K. Hasunuma K. Mol. Gen. Genet. 1997; 256: 593-601Crossref PubMed Scopus (28) Google Scholar), suggesting that phosphorylation of NDK-1 is critical for the photoresponse. NDK is a ubiquitous enzyme that catalyzes the transfer of the γ-phosphate group of nucleoside 5′-triphosphate to nucleoside 5′-diphosphate to form nucleoside 5′-triphosphate (28Parks, R. E., Jr., Agarwal, R. P., The Enzymes, Boyer, P. D., 1973, 307, 333, Academic Press, NY.Google Scholar). Recent studies suggest that NDK is involved in various signal transduction pathways such as suppression of tumor metastasis (29Steeg, P. S., Bevilacqua, G., Pozzatti, R., Liotta, L. A., Sobel, M. E., Cancer Res., 48, 1988, 6550, 6554.Google Scholar), modulation of muscarinic K+ channels (30Otero A.S. Doyle M.B. Hartsough M.T. Steeg P.S. Biochim. Biophys. Acta. 1999; 1449: 157-168Crossref PubMed Scopus (18) Google Scholar), transcriptional activation of proto-oncogenes (31Postel E.H. Berberich S.J. Flint S.J. Ferrone C.A. Science. 1993; 261: 478-480Crossref PubMed Scopus (482) Google Scholar), development of the wing disc cells inDrosophila melanogaster (32Biggs J. Hersperger E. Steeg P.S. Liotta L.A. Shearn A. Cell. 1990; 63: 933-940Abstract Full Text PDF PubMed Scopus (333) Google Scholar), and sexual development inSchizosaccharomyces pombe (33Izumiya H. Yamamoto M. J. Biol. Chem. 1995; 270: 27859-27864Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). In plants, the phosphorylation of NDK in pea, PNDK1, was stimulated by red light, and the stimulation was reversed by subsequent irradiation with far-red light (34Tanaka N. Ogura T. Noguchi T. Hirano H. Yabe N. Hasunuma K. J. Photochem. Photobiol. 1998; 45: 113-121Crossref PubMed Scopus (42) Google Scholar). PNDK1 also showed protein-phosphorylation activity (35Ogura T. Tanaka N. Yabe N. Komatsu S. Hasunuma K. Photochem. Photobiol. 1999; 69: 397-403Crossref Google Scholar). InA. thaliana, NDPK2 is hypothesized to be a photo-signaling component because of the partial defect in photo-dependent cotyledon opening and greening in the T-DNA insertional disruptant of NDPK2 (36Choi G. Yi H. Lee J. Kwon Y.-K. Soh M.S. Shin B. Luka Z. Hahn T.-R. Song P.-S. Nature ( London ). 1999; 401: 610-613Crossref PubMed Scopus (254) Google Scholar). Several roles for NDK in signal transduction have been proposed: transcriptional activation (31Postel E.H. Berberich S.J. Flint S.J. Ferrone C.A. Science. 1993; 261: 478-480Crossref PubMed Scopus (482) Google Scholar, 37Zimmermann S. Baumann A. Jaekel K. Marbach I. Engelberg D. Frohnmeyer H. J. Biol. Chem. 1999; 274: 17017-17024Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), supply of GTP to G-proteins (38Kimura N. Shimada N. J. Biol. Chem. 1983; 258: 2278-2283Abstract Full Text PDF PubMed Google Scholar), regulation of muscarinic K+ channels (30Otero A.S. Doyle M.B. Hartsough M.T. Steeg P.S. Biochim. Biophys. Acta. 1999; 1449: 157-168Crossref PubMed Scopus (18) Google Scholar), interaction with other signaling proteins (36Choi G. Yi H. Lee J. Kwon Y.-K. Soh M.S. Shin B. Luka Z. Hahn T.-R. Song P.-S. Nature ( London ). 1999; 401: 610-613Crossref PubMed Scopus (254) Google Scholar, 39Leung S.-M. Hightower L.E. J. Biol. Chem. 1997; 272: 2607-2614Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar), and phosphorylation of proteins (40Freije J.M.P. Blay P. MacDonald N.J. Manrow R.E. Steeg P.S. J. Biol. Chem. 1997; 272: 5525-5532Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). NDK-1 has been shown to be able to phosphorylate itself and other proteins (27Ogura Y. Yoshida Y. Ichimura K. Aoyagi C. Yabe N. Hasunuma K. Eur. J. Biochem. 1999; 266: 709-714Crossref PubMed Scopus (21) Google Scholar). Based on its protein-phosphorylation activity, NDK-1 may be involved in phosphorelay signaling. In the present study, we have investigated the roles of NDK-1 in the light signal transduction pathway through the activity from nucleoside 5′-triphosphate to nucleoside 5′-diphosphate and the phosphotransfer activity to other The N. crassa wild type and from the The psp A a ndk-1P72H in this (17Oda K. Hasunuma K. Mol. Gen. Genet. 1997; 256: 593-601Crossref PubMed Scopus (28) Google Scholar). was in with 2‥ of ndk-1P72H and the developed in on for and with the in in a with a and from the a for and through N. crassa wild type and ndk-1P72H for The mycelia by and with a and in was by the of R. M. Russo V.E.A. Genet. 1990; Scholar). of was by gel electrophoresis and to a of membrane with ndk-1 was in and 1 by using for in 1 of on and by in of protein and was as after for of the of protein was by gel electrophoresis and a of The membrane was with in using purified with a the protein phosphorylation the was with for in and (17Oda K. Hasunuma K. Mol. Gen. Genet. 1997; 256: 593-601Crossref PubMed Scopus (28) Google Scholar). The of from the DNA of the of the and of the ndk-1 as and from wild type as well aspsp A was with 1 of and of in a reaction and for for 1 The was with a reaction of of and of reverse and for 1 to cDNA of specifically by with of and using a reaction in a of and of was and 72 for cycles with a of 72 the of and by the chain N. crassa ndk-1P72H a was with a ndk-1 genomic DNA or a for ectopic expression of ndk-1 cDNA to and S.J. C. S. A. PubMed Scopus Google Scholar). was by of a genomic DNA ndk-1 which the as a M.L. Genet. 1993; Scholar). ectopic expression of cDNA in N. crassa, we a which for and the and for ectopic expression Mol. Gen. Genet. PubMed Scopus Google Scholar). This has a the and for and The ndk-1 and was to The on 2‥ 2‥ and 1 which was to have a the and to the and for of ndk-1 of and and which a glutathioneS-transferase in the of a and S. pombe was with or in S. A. P. 1991; PubMed Scopus Google Scholar) for and the was in an of and of was and the cells by cycles of a of 1 of and 1 of on the was with and for The was with protein was with an of and The protein was from the of after electrophoresis by the The for NDK activity determined by a to J. PubMed Scopus Google Scholar) In this of purified recombinant protein was The of and The from the The activity of and to phosphorylate myelin basic protein was to a (27Ogura Y. Yoshida Y. Ichimura K. Aoyagi C. Yabe N. Hasunuma K. Eur. J. Biochem. 1999; 266: 709-714Crossref PubMed Scopus (21) Google Scholar). The of or or 1 and was for 1 after of of a of the a part of the gel was The activity of this of gel was by the with a The autophosphorylation activity of or was by of of the protein in the reaction The reaction was by on and after the reaction was by The was the as phosphorylation of NDK-1 was in the from the mutant psp (see A and K. Hasunuma K. Mol. Gen. Genet. 1997; 256: 593-601Crossref PubMed Scopus (28) Google Scholar). This suggested that the of ndk-1 was to the accumulation of or to the protein We the levels of ndk-1 mRNA and NDK-1 protein in psp blot analysis showed no in the accumulation ofndk-1 mRNA psp(ndk-1P72H) and wild type 1 In Western blot analysis using indicated that about 25‥ of NDK-1 protein was in psp(ndk-1P72H) compared with that in wild type 1 from The of NDK-1 proteins from wild type and mutant by the of the Western these we that an acid substitution in NDK-1 itself affected its or this the of ndk-1 which was by was in the and of ndk-1 cDNA as for A to the was 1 from wild type and psp(ndk-1P72H) A and In the psp in The was in the the was and the was a of with the of proline 72 with the of cDNA from wild type was to that of A was also by The of this was to that of ndk-1 except that the from the to base from the not The acid of this was to that of ndk-1 except that it in the This was as The that of the psp mutant not suggested that was the of The of this because the was compared with that ofndk-1 1 and no was by Western blot analysis. the mutation was with the caused the phenotype of a genomic DNA wild type ndk-1 or for cDNA ectopic expression was by of on a The of the was by blot analysis not the genomic DNA ofndk-1 and and the and The of NDK-1 in the was by Western blot analysis. The able to as NDK-1 as the wild type Phosphorylation of NDK-1 was also The to phosphorylate NDK-1 itself was in all of the In NDK-1 was in psp(ndk-1P72H) but in other and the wild type These results and the results in Fig. A and C, showed that the mutation in ndk-1 caused NDK-1 accumulation of about 25‥ and no activity of NDK-1 phosphorylation in psp compared with those in wild type The perithecial of the ndk-1 The that the beaks form the or other position on the are shown in In the of the perithecial beak was of the as a protoperithecial white light was from to the of the the beaks formed the of the in the wild the of the beaks was white light was from as in In psp(ndk-1P72H) wild beaks formed the of the Fig. shows of wild type, and in or These results that the phenotype of the psp mutant is caused by mutation and that NDK-1 a in the light-responsive regulation of perithecial polarity. mutation was determined to polarity of wild P72H and the light was to the of the in the and through a wild for NDK activity and protein-phosphorylation activity of NDK-1 or NDK-1P72H determined using purified recombinant and showed activity for and in the not was as a of activity to the for the of shown in the that to as a and the and of protein with and and of protein with respectively. results also by of and These findings indicated that the activity of NDK-1 was not affected by the P72H In the autophosphorylation and protein kinase of greatly compared with those of not However, it is difficult to this in because the of and is this we protein is from that of and V of proteins of NDK-1 and NDK-1P72H for activity for of with V is the of the enzyme in a The of with V is the of the enzyme of proteins was by of proteins by Western was about 5‥ of compared with that in This that the of NDK-1P72H to phosphorylate itself was greatly the activity to phosphorylate was proteins with and various of or and by shows that is with The determined by the of the of and the activity of and of protein with and and of protein with respectively. The V with was about 2‥ of that with These results revealed that the P72H mutation affected the of NDK-1 to phosphorylate both itself and and V of proteins of NDK-1 and NDK-1P72H for phosphotransfer activity for of with in a The of with We that the P72H substitution in NDK-1 partial reduction in accumulation of NDK-1 and of the activity to phosphorylate itself and This proline is the NDK genes to (27Ogura Y. Yoshida Y. Ichimura K. Aoyagi C. Yabe N. Hasunuma K. Eur. J. Biochem. 1999; 266: 709-714Crossref PubMed Scopus (21) Google Scholar) and is to be the and the from of NDK in C. I. S. P. R. M. J. EMBO J. PubMed Scopus Google Scholar, S. G. I. M.L. M. J. J. Mol. Biol. 1994; PubMed Scopus Google Scholar). These in light for perithecial polarity. In of either wild genomic DNA or NDK-1 was and as in the wild type, in the NDK-1 was in the wild type or other However, the of NDK-1 in was because was no in the phosphorylation of NDK-1 compared with the wild type and other This may be caused by the position of the in the N. crassa The of NDK-1 accumulation and phosphorylation in the in results in the normal light of perithecial polarity that is defective in This shows that the was an allele of the ndk-1 and indicated that NDK-1 a in the regulation of perithecial polarity in to WC-1 and are the in the blue light signaling pathway in N. crassa (21Ballario P. Vittorioso P. Margrelli A. Talora C. Cabibbo A. Macino G. EMBO J. 1996; 15: 1650-1657Crossref PubMed Scopus (375) Google Scholar, 22Linden H. Macino G. EMBO J. 1997; 16: 98-109Crossref PubMed Scopus (286) Google Scholar). are transcription and form a that was suggested to function as a signal (23Ballario P. Talora C. Galli D. Linden H. Macino G. Mol. Microbiol. 1998; 29: 719-729Crossref PubMed Scopus (150) Google Scholar). The WC is also proposed to be a on its in the LOV domain with Arabidopsis blue light receptor phototropin (7Briggs W.R. Huala E. Annu. Rev. Cell Dev. Biol. 1999; 15: 33-62Crossref PubMed Scopus (332) Google Scholar). WC-1 is and this phosphorylation in to light (24Talora C. Franchi L. Linden H. Ballario P. Macino G. EMBO J. 1999; 18: 4961-4968Crossref PubMed Scopus (197) Google Scholar). This the of a protein phosphorylation in the blue light signaling pathway. However, the protein that the light signaling have not been identified in N. crassa, protein kinase is to a (25Arpaia G. Cerri F. Baima S. Macino G. Mol. Gen. Genet. 1999; 262: 314-322Crossref PubMed Scopus (58) Google Scholar). the increase in the of NDK-1 phosphorylation after blue light irradiation is but not in the protein-phosphorylation activity of NDK-1 to the of the light signaling downstream of WC of perithecial polarity was also defective in wc-1 and other light-insensitive in such as phototropism of perithecial not This that various by pathways downstream of WC proteins and that NDK-1 was involved in such pathway perithecial polarity. was no in the of accumulation wild type but Western analysis revealed that about 25‥ of NDK-1P72H protein was in the compared with that of wild This that regulation an for NDK-1 In a NDK was with as an protein S.-M. Hightower L.E. J. Biol. Chem. 1997; 272: 2607-2614Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). be that the the proline 72 was responsible for the with proteins such as the or of We are on proteins with NDK-1 by and by The reduction in V for the phosphorylation of by shows that the P72H substitution not autophosphorylation but also protein kinase of This that autophosphorylation and protein kinase from that of activity of the the proline 72 is proposed to to the to the the decrease in the NDK-1 protein kinase activity, caused by protein accumulation and V in the activity, Neurospora to the of to We that NDK-1 a in an pathway autophosphorylation and protein kinase that regulate perithecial polarity in to The activity of NDK-1P72H was in of autophosphorylation and protein kinase but not the of NDK-1 in signal transduction GTP to ion channels (30Otero A.S. Doyle M.B. Hartsough M.T. Steeg P.S. Biochim. Biophys. Acta. 1999; 1449: 157-168Crossref PubMed Scopus (18) Google Scholar), may not be affected by the However, we that GTP to NDK-1 also signal through the autophosphorylation and protein kinase activities, that may be a signal transduction pathway by these of of the of NDK-1 in the signaling pathway may of the proteins that with NDK-1, such as a protein the NDK-1 and a protein by We are to D. D. for critical and to R. Briggs for critical and of the We C. N. K. and K. Ichimura for C. Aoyagi for in H. C. and S. for in Neurospora and S. for