- Email: [email protected]
Molecular cloning and characterisation of a mouse gene encoding an isoform of the neuronal cyclin-dependent kinase 5 (CDK5) activator
Biochimica et Biophysica Acta 1398 (1998) 371^376
Short sequence-paper
Molecular cloning and characterisation of a mouse gene encoding an isoform of the neuronal cyclin-dependent kinase 5 (CDK5) activator Fredrik Nilde¨n, Anders Ba«ckstro«m, Christina Bark * Department of Developmental Neuroscience, Uppsala University, BMC, Box 587, 751 23 Uppsala, Sweden Received 31 March 1998; accepted 14 April 1998
Abstract We have isolated and characterised the mouse gene for the p39 activator, designated p39is, which encodes a protein of 369 amino acids. The mouse p39 protein exhibits 95% amino acid identity to a previously characterised human p39 cDNA and the novel gene sequence corresponds to a single genomic locus in mouse. The p39is mRNA is highly expressed in the mouse central nervous system. ß 1998 Elsevier Science B.V. All rights reserved. Keywords: CDK5 activator; Cdc2-like protein kinase; Neuronal protein; p35; p39; Gene
Cyclin-dependent kinases (CDKs) are serine/threonine kinases ranging in molecular mass between 33 and 40 kDa and well known to be involved in cell cycle control [1]. Cyclins are a family of structurally related proteins originally de¢ned as proteins whose levels oscillate during the cell division cycle [2]. The active CDK thus exists as a heterodimer of a CDK unit (CDK1^CDK8) and a cyclin unit (cyclins A^I) in a variety of combinations [3]. Not all CDKs participate in events strictly related to cell-cycle control [4]. CDK5 was isolated due to its homology to cdc2, but showed distinct di¡erences in expression and activation patterns [5]. Neuronal cdc2-like kinase, originally puri¢ed from bovine brain [6], is a heterodimer of CDK5 and neuronal-speci¢c activating subunits. Two distinct subunits have so far been described, p35 Abbreviations: bp, base pair(s); CDK, cyclin-dependent kinase; kb, kilobase(s); MOPS, 4-morpholinepropanesulphonic acid; SDS, sodium dodecyl sulphate; SSC, saline sodium citrate * Corresponding author. Fax: +46 (18) 559017; E-mail: [email protected]
[7,8] and p39 [9], which both display limited homology with classical cyclins. In this study, we describe the cloning and characterisation of a novel mouse gene, designated p39is, encoding an isoform belonging to the CDK5 activator family with an expression pattern limited within the central nervous system. A VFIX II mouse 129/SVJ genomic library (Stratagene) was screened using a 5P-[K-32 P]dCTP randomly labelled (RediPrime, Amersham) human p35 PCR fragment as a probe. Recombinant phage DNA was puri¢ed, and characterised with restriction enzyme cleavages and Southern blots essentially as described [10]. All subclones were sequenced on both strands using a Perkin-Elmer 373 automated sequencer (Applied Biosystems) and analysed using the GCG package (Wisconsin Computer Group). Mouse chromosomal DNA was prepared from ES cells (129/SVJ) using standard procedures [10]. Genomic DNA, 12 Wg per digestion, was cleaved with the enzyme of choice and separated on 1.0% (w/v) agarose gel together with DNA markers (kb ladder and V HindIII marker, Gibco BRL). Extraction of
0167-4781 / 98 / $19.00 ß 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 9 8 ) 0 0 0 7 3 - 6
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total RNA from various tissues from adult mice (female C57BL) was performed using the guanidium thiocyanate/CsTFA method (Pharmacia; [10]). 20 Wg of total RNA/slot was separated on formaldehyde/1% (w/v) agarose gels, transferred to nylon membranes (Hybond N+, Amersham) and hybridised with a 700 bp probe from the p39is gene (see Fig. 1). The screening of the mouse genomic library uncovered 16 distinct recombinant phages with varying hybridisation intensity. Several DNA fragments, hybridising to the p35 PCR probe, were subcloned into plasmid vectors prior nucleotide sequence analysis. Nucleotide sequence analysis of subclones from one phage revealed the presence of a novel gene. The gene was designated p39is, dependent on the sequence similarity to a human p39 cDNA sequence previously reported [9]. A restriction enzyme map was established of the insert of 14.6 kb (Fig. 1A and B). To determine whether the restriction map of the p39is phage insert showed an identical restriction enzyme cleavage pattern compared to chromosomal mouse DNA and to investigate whether the cloned gene corresponded to a single genomic locus, Southern blots were performed (Fig. 1C). The probe used was a PstI fragment of approx. 700 bp from the p39is gene (see Fig. 1B and C for details). The results showed that the sizes of hybridising fragments correlated between the phage map and the mouse chromosomal DNA on the genomic blot and it could be concluded that no DNA rearrangements had occurred during propagation of the recombinant phage and that p39is corresponded to a single-copy gene. To further establish that only one locus corresponded to p39is sequences in genomic DNA, a chromosomal blot was hybridised with a 1550 bp NcoI-XhoI fragment, spanning 500 bp upstream from the start codon and 1050 bp of the coding sequence (see Fig. 1B for details). This con¢rmed that a single genomic locus corresponded to p39is sequences in mouse (data not shown). Nucleotide sequence analysis of two overlapping subclones, a KpnI fragment spanning 4 kb and a BglII fragment spanning 1.6 kb, from the S1 phage revealed that the open reading frame of the p39is protein was contained within one exon (Fig. 2). In total, 4521 bp of genomic DNA was sequenced, spanning the entire p39is open reading frame and £anking sequences (see
Fig. 2 for details). The open reading frame consists of 1110 bp, with a putative start codon characteristic of being e¤ciently used for translation, according to Kozak's rule [11] and a putative polyadenylation signal was detected in the 3P-untranslated region. The sequence within the open reading frame displayed 97% nucleotide sequence identity with the known cDNA for human p39 [9] and 95% identity at the amino acid level. The deduced amino acid sequence encodes a protein comprising of 369 amino acids, with an insert of two extra serines at positions 351^ 352 compared to the human p39 cDNA (Fig. 3). A notable feature of the p39is gene is the absence of introns interrupting the coding region, much in the same manner as reported for the gene encoding p35 [12]. Northern blot hybridisations were performed on a variety of tissues from adult mice (C57BL). The probe used was the same as for the genomic blots, a PstI fragment spanning amino acid residues 250^ 369 of the p39 protein and part of the 3P-untranslated region of the p39is gene. RNA transcripts of approx. 2.6 kb in size were detected in lanes containing mouse whole brain, hippocampus, cerebellum and spinal cord (Fig. 4). No peripheral tissue examined showed any evident expression of the p39is mRNA. Based on structural similarity, both on the nucleotide and amino acid level, and the restricted mRNA expression in neural tissue, we suggest that this is the mouse counterpart for the human p39 cDNA previously reported [9]. The p39is gene, which is the ¢rst p39 gene isolated for the novel family of CDK5 activator proteins, is a single-copy gene in mouse. The p39is gene shares structural features with the p35 gene in the respect that the open reading frame of the protein is encoded within one exon. The high degree of similarity between p35 and p39 amino acid sequences, together with the analogous gene organisation thus might suggest that they both have arisen from a common ancestor gene. The expression of p35 and p39 mRNA in various cell lines implies that their biological activities are di¡erent [9]. CDK5 and p35 are highly conserved among species but p39/p39is exhibits less identity between mouse and human. Still, from results recently described, p39/p39is is conserved in the protein domains which corresponds to CDK5 binding and activation in the p35 polypeptide sequence [13]. Fig. 3 shows the comparison between mouse p39is, human p39 and mouse
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373
Fig. 1. Genomic restriction map of the p39is gene. (A) A restriction enzyme map illustrating the recombinant phage spanning the p39is gene in mouse. The insert in the recombinant phage spans 14.6 kb of DNA. The position of the open reading frame within the transcribed region of the p39is gene is indicated as a dark box. *Not all restriction sites for this enzyme are shown. (B) A total of 4521 bp of genomic DNA was sequenced, spanning 5P and 3P £anking regions of the coding sequence. The entire open reading frame is contained within one exon as indicated with a dark box. The 720 bp PstI fragment used as a p39is speci¢c probe in Northern hybridisation and genomic blots is indicated. (C) Southern blot with mouse chromosomal DNA. The genomic blot con¢rms that the p39is gene is a single-copy gene. The hybridising KpnI fragment of 4 kb, a BglII fragment of 1.6 kb, an XbaI fragment of 8 kb and an approx. 0.7 kb PstI fragment all correlate with the expected sizes from the gene map.
p35 amino acid sequences. Within the domains, proposed to be functionally important, the amino acid identity extends 90% between the three proteins. Of particular interest is the C-terminal region of the p35 protein since it comprises the substrate speci¢city of the CDK5/p35 kinase [14]. Indeed, there are data which suggest that the optimal substrate speci¢city for the p35/CDK5 kinase is a ¢xed serine-proline motif, with a consensus reading KHHKSPKHR [15]. P39is shows an insert of 27 amino acids in the C-terminal region and it is intriguing to speculate that this might alter the substrate speci¢city of the
CDK5 complex. However, it is noteworthy that in the human p39 homologue the insertion is only 25 amino acids [9]. The extra amino acids inserted in the carboxyl terminus of the p39/p39is proteins share similarity in two domains corresponding to amino acids 329^341 and 349^369, respectively, as calculated from the mouse polypeptide sequence (Fig. 3). A possibility is that CDK5/p35 and CDK5/p39is exhibit di¡erent substrate preferences and that might be found within one of these two domains. Two regions of the amino terminal of the p39is protein might also be of speci¢c importance. Amino acids
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Fig. 2. Nucleotide sequence of the mouse p39is gene. Nucleotide sequence and deduced amino acid sequence of the mouse gene for the CDK5 activator p39is. The open reading frame spans 1110 bp and encodes a protein of 369 amino acids. The ¢rst nucleotide of the ATG start codon is designated +1 and the deduced amino acid sequence is presented below the open reading frame. Putative regulatory cis elements were found by computer analysis approx. 200 bp upstream from the start codon and included binding sites for AP2 and NGF1A. These recognition sequences have also been identi¢ed by computer analysis in the promotor region of the p35 gene [12]. Also underlined are CA and GA repeats present upstream from the gene. A potential polyadenylation signal is found at positions 2318^2323, approx. 1500 bp downstream from the stop codon. The GenBank accession number for the mouse p39is gene is U90267.
10^53 are, except for a conservative valine to alanine substitution between p39 and p39is, conserved be-
tween the mouse and human proteins and show only limited homology to p35 (Fig. 3). In addition,
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375
Fig. 3. Amino acid sequence alignment of p39, p39is and p35 proteins. Amino acid comparison between the deduced amino acid sequences of human p39 and mouse p39is and mouse p35. The p39 protein consists of 367 amino acids and the p39is polypeptide of 369 residues exhibits 95% identity to the human counterpart. The 307 amino acid long p35 protein displays 57% identity to the human p39is sequence. Shaded areas indicate conserved amino acids between the three proteins. The two underlined sequences show regions of the p35 proteins implied to correspond to the binding and activating domain for CDK5, respectively.
amino acids 86^173 seem to be unique for the p39is/ p39 activator variant (Fig. 3). Possibly, CDK5/p35 and CDK5/p39is exhibit di¡erent substrate preferences. The CDK5/p35 complex has been shown to promote neurite extension of cultured cortical neurons, which agrees with the proposed role of neuronal CDK5 activity in the control of cytoskeletal functions [16]. A targeted disruption of the gene for CDK5 results in abnormal brain development and perinatal death, data which show the importance of CDK5 during brain development [17]. Also, mice
lacking p35 display cortical lamination defects, seizures and adult lethality [18]. Whether a targeted disruption of the p39is gene also results in developmental defects remains to be seen. Thus, several studies have reported the importance of this novel family of kinase complexes during nervous system development. Future studies will provide answers regarding substrate speci¢city and functional roles for this family of kinases in the mature nervous system. This work was supported by the Swedish Natural Research Council, Grants B-AA/BU 09714-311, Axel
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References
Fig. 4. Tissue distribution of p39is mRNA. Northern blot showing that the mRNA expression of p39is is neural speci¢c. 20 Wg of total RNA was loaded in each lane. (A) Hybridisation with the p39is speci¢c probe shows high expression of p39is in total brain and cerebellum and moderate levels in the hippocampus and spinal cord. None of the peripheral tissues examined displayed any evident p39is mRNA expression. The size of the transcript was estimated to be approx. 2.6 kb, compared to 18S and 28S ribosomal RNA visualised by ethidium bromide staining. (B) Hybridisation with a GADPH fragment was performed to verify proper transfer to the membrane and to ensure that the level of total RNA loaded/slot were comparable.
and Signe Lagermans Foundation, Pharmacia Research Foundation and the Alzheimer Foundation Sweden (to C.B.). We are grateful to Helena Vretman for skilful technical assistance in DNA sequencing and we thank Dr. Alexander Mercer for helpful revision of the manuscript.
[1] C. Norbury, P. Nurse, Annu. Rev. Biochem. 61 (1992) 441^ 470. [2] J. Pines, Semin. Cell. Biol. 5 (1994) 399^408. [3] M. Doree, S. Galas, FASEB J. 8 (1994) 1114^1121. [4] R.P. Fisher, Curr. Opin. Genet. Dev. 7 (1997) 32^38. [5] L.H. Tsai, T. Takahashi, V.J. Caviness, E. Harlow, Development 119 (1993) 1029^1040. [6] J. Lew, K. Beaudette, C.M. Litwin, J.H. Wang, J. Biol. Chem. 267 (1992) 13383^13390. [7] J. Lew, Q.-Q. Huang, Z. Qi, R.J. Winkfein, R. Aebersold, T. Hunt, J.H. Wang, Nature 371 (1994) 423^426. [8] L.H. Tsai, I. Delalle, V.S. Caviness Jr., T. Chae, E. Harlow, Nature 371 (1994) 419^423. [9] D. Tang, J. Yeung, K.Y. Lee, M. Matsushita, H. Matsui, K. Tomizawa, O. Hatase, J.H. Wang, J. Biol. Chem. 270 (1995) 26897^26903. [10] J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning : a Laboratory Manual, 2nd edn., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989, pp. 1.42^1.46. [11] M. Kozak, Cell 44 (1986) 283^292. [12] T. Ohshima, C.A. Kozak, J.W. Nagle, H.C. Pant, R.O. Brady, A.B. Kulkarni, Genomics 35 (1996) 372^375. [13] R.Y.C. Poon, J. Lew, T. Hunter, J. Biol. Chem. 272 (1997) 5703^5708. [14] D.M. Tang, A.C.S. Chun, M.J. Zhang, J.H. Wang, J. Biol. Chem. 272 (1997) 12318^12327. [15] Z. Songyang, K.P. Lu, Y.T. Kwon, L.H. Tsai, O. Filhol, C. Cochet, D.A. Brickey, T.R. Soderling, C. Bartleson, D.J. Graves, A.J. DeMaggio, M.F. Hoekstra, J. Blenis, T. Hunter, L.C. Cantley, Mol. Cell. Biol. 16 (1996) 6486^6493. [16] M. Nikolic, H. Dudek, Y.T. Kwon, Y.F. Ramos, L.H. Tsai, Genes Dev. 10 (1996) 816^825. [17] T. Ohshima, J.M. Ward, C.G. Huh, G. Longenecker, H.C. Veeranna Pant, R.O. Brady, L.J. Martin, A.B. Kulkarni, Proc. Natl. Acad. Sci. USA 93 (1996) 11173^11178. [18] T. Chae, Y.T. Kwon, R. Bronson, P. Dikkes, E. Li, L.-H. Tsai, Neuron 18 (1997) 29^42.
BBAEXP 91143 1-7-98
Short sequence-paper
Molecular cloning and characterisation of a mouse gene encoding an isoform of the neuronal cyclin-dependent kinase 5 (CDK5) activator Fredrik Nilde¨n, Anders Ba«ckstro«m, Christina Bark * Department of Developmental Neuroscience, Uppsala University, BMC, Box 587, 751 23 Uppsala, Sweden Received 31 March 1998; accepted 14 April 1998
Abstract We have isolated and characterised the mouse gene for the p39 activator, designated p39is, which encodes a protein of 369 amino acids. The mouse p39 protein exhibits 95% amino acid identity to a previously characterised human p39 cDNA and the novel gene sequence corresponds to a single genomic locus in mouse. The p39is mRNA is highly expressed in the mouse central nervous system. ß 1998 Elsevier Science B.V. All rights reserved. Keywords: CDK5 activator; Cdc2-like protein kinase; Neuronal protein; p35; p39; Gene
Cyclin-dependent kinases (CDKs) are serine/threonine kinases ranging in molecular mass between 33 and 40 kDa and well known to be involved in cell cycle control [1]. Cyclins are a family of structurally related proteins originally de¢ned as proteins whose levels oscillate during the cell division cycle [2]. The active CDK thus exists as a heterodimer of a CDK unit (CDK1^CDK8) and a cyclin unit (cyclins A^I) in a variety of combinations [3]. Not all CDKs participate in events strictly related to cell-cycle control [4]. CDK5 was isolated due to its homology to cdc2, but showed distinct di¡erences in expression and activation patterns [5]. Neuronal cdc2-like kinase, originally puri¢ed from bovine brain [6], is a heterodimer of CDK5 and neuronal-speci¢c activating subunits. Two distinct subunits have so far been described, p35 Abbreviations: bp, base pair(s); CDK, cyclin-dependent kinase; kb, kilobase(s); MOPS, 4-morpholinepropanesulphonic acid; SDS, sodium dodecyl sulphate; SSC, saline sodium citrate * Corresponding author. Fax: +46 (18) 559017; E-mail: [email protected]
[7,8] and p39 [9], which both display limited homology with classical cyclins. In this study, we describe the cloning and characterisation of a novel mouse gene, designated p39is, encoding an isoform belonging to the CDK5 activator family with an expression pattern limited within the central nervous system. A VFIX II mouse 129/SVJ genomic library (Stratagene) was screened using a 5P-[K-32 P]dCTP randomly labelled (RediPrime, Amersham) human p35 PCR fragment as a probe. Recombinant phage DNA was puri¢ed, and characterised with restriction enzyme cleavages and Southern blots essentially as described [10]. All subclones were sequenced on both strands using a Perkin-Elmer 373 automated sequencer (Applied Biosystems) and analysed using the GCG package (Wisconsin Computer Group). Mouse chromosomal DNA was prepared from ES cells (129/SVJ) using standard procedures [10]. Genomic DNA, 12 Wg per digestion, was cleaved with the enzyme of choice and separated on 1.0% (w/v) agarose gel together with DNA markers (kb ladder and V HindIII marker, Gibco BRL). Extraction of
0167-4781 / 98 / $19.00 ß 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 9 8 ) 0 0 0 7 3 - 6
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total RNA from various tissues from adult mice (female C57BL) was performed using the guanidium thiocyanate/CsTFA method (Pharmacia; [10]). 20 Wg of total RNA/slot was separated on formaldehyde/1% (w/v) agarose gels, transferred to nylon membranes (Hybond N+, Amersham) and hybridised with a 700 bp probe from the p39is gene (see Fig. 1). The screening of the mouse genomic library uncovered 16 distinct recombinant phages with varying hybridisation intensity. Several DNA fragments, hybridising to the p35 PCR probe, were subcloned into plasmid vectors prior nucleotide sequence analysis. Nucleotide sequence analysis of subclones from one phage revealed the presence of a novel gene. The gene was designated p39is, dependent on the sequence similarity to a human p39 cDNA sequence previously reported [9]. A restriction enzyme map was established of the insert of 14.6 kb (Fig. 1A and B). To determine whether the restriction map of the p39is phage insert showed an identical restriction enzyme cleavage pattern compared to chromosomal mouse DNA and to investigate whether the cloned gene corresponded to a single genomic locus, Southern blots were performed (Fig. 1C). The probe used was a PstI fragment of approx. 700 bp from the p39is gene (see Fig. 1B and C for details). The results showed that the sizes of hybridising fragments correlated between the phage map and the mouse chromosomal DNA on the genomic blot and it could be concluded that no DNA rearrangements had occurred during propagation of the recombinant phage and that p39is corresponded to a single-copy gene. To further establish that only one locus corresponded to p39is sequences in genomic DNA, a chromosomal blot was hybridised with a 1550 bp NcoI-XhoI fragment, spanning 500 bp upstream from the start codon and 1050 bp of the coding sequence (see Fig. 1B for details). This con¢rmed that a single genomic locus corresponded to p39is sequences in mouse (data not shown). Nucleotide sequence analysis of two overlapping subclones, a KpnI fragment spanning 4 kb and a BglII fragment spanning 1.6 kb, from the S1 phage revealed that the open reading frame of the p39is protein was contained within one exon (Fig. 2). In total, 4521 bp of genomic DNA was sequenced, spanning the entire p39is open reading frame and £anking sequences (see
Fig. 2 for details). The open reading frame consists of 1110 bp, with a putative start codon characteristic of being e¤ciently used for translation, according to Kozak's rule [11] and a putative polyadenylation signal was detected in the 3P-untranslated region. The sequence within the open reading frame displayed 97% nucleotide sequence identity with the known cDNA for human p39 [9] and 95% identity at the amino acid level. The deduced amino acid sequence encodes a protein comprising of 369 amino acids, with an insert of two extra serines at positions 351^ 352 compared to the human p39 cDNA (Fig. 3). A notable feature of the p39is gene is the absence of introns interrupting the coding region, much in the same manner as reported for the gene encoding p35 [12]. Northern blot hybridisations were performed on a variety of tissues from adult mice (C57BL). The probe used was the same as for the genomic blots, a PstI fragment spanning amino acid residues 250^ 369 of the p39 protein and part of the 3P-untranslated region of the p39is gene. RNA transcripts of approx. 2.6 kb in size were detected in lanes containing mouse whole brain, hippocampus, cerebellum and spinal cord (Fig. 4). No peripheral tissue examined showed any evident expression of the p39is mRNA. Based on structural similarity, both on the nucleotide and amino acid level, and the restricted mRNA expression in neural tissue, we suggest that this is the mouse counterpart for the human p39 cDNA previously reported [9]. The p39is gene, which is the ¢rst p39 gene isolated for the novel family of CDK5 activator proteins, is a single-copy gene in mouse. The p39is gene shares structural features with the p35 gene in the respect that the open reading frame of the protein is encoded within one exon. The high degree of similarity between p35 and p39 amino acid sequences, together with the analogous gene organisation thus might suggest that they both have arisen from a common ancestor gene. The expression of p35 and p39 mRNA in various cell lines implies that their biological activities are di¡erent [9]. CDK5 and p35 are highly conserved among species but p39/p39is exhibits less identity between mouse and human. Still, from results recently described, p39/p39is is conserved in the protein domains which corresponds to CDK5 binding and activation in the p35 polypeptide sequence [13]. Fig. 3 shows the comparison between mouse p39is, human p39 and mouse
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373
Fig. 1. Genomic restriction map of the p39is gene. (A) A restriction enzyme map illustrating the recombinant phage spanning the p39is gene in mouse. The insert in the recombinant phage spans 14.6 kb of DNA. The position of the open reading frame within the transcribed region of the p39is gene is indicated as a dark box. *Not all restriction sites for this enzyme are shown. (B) A total of 4521 bp of genomic DNA was sequenced, spanning 5P and 3P £anking regions of the coding sequence. The entire open reading frame is contained within one exon as indicated with a dark box. The 720 bp PstI fragment used as a p39is speci¢c probe in Northern hybridisation and genomic blots is indicated. (C) Southern blot with mouse chromosomal DNA. The genomic blot con¢rms that the p39is gene is a single-copy gene. The hybridising KpnI fragment of 4 kb, a BglII fragment of 1.6 kb, an XbaI fragment of 8 kb and an approx. 0.7 kb PstI fragment all correlate with the expected sizes from the gene map.
p35 amino acid sequences. Within the domains, proposed to be functionally important, the amino acid identity extends 90% between the three proteins. Of particular interest is the C-terminal region of the p35 protein since it comprises the substrate speci¢city of the CDK5/p35 kinase [14]. Indeed, there are data which suggest that the optimal substrate speci¢city for the p35/CDK5 kinase is a ¢xed serine-proline motif, with a consensus reading KHHKSPKHR [15]. P39is shows an insert of 27 amino acids in the C-terminal region and it is intriguing to speculate that this might alter the substrate speci¢city of the
CDK5 complex. However, it is noteworthy that in the human p39 homologue the insertion is only 25 amino acids [9]. The extra amino acids inserted in the carboxyl terminus of the p39/p39is proteins share similarity in two domains corresponding to amino acids 329^341 and 349^369, respectively, as calculated from the mouse polypeptide sequence (Fig. 3). A possibility is that CDK5/p35 and CDK5/p39is exhibit di¡erent substrate preferences and that might be found within one of these two domains. Two regions of the amino terminal of the p39is protein might also be of speci¢c importance. Amino acids
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Fig. 2. Nucleotide sequence of the mouse p39is gene. Nucleotide sequence and deduced amino acid sequence of the mouse gene for the CDK5 activator p39is. The open reading frame spans 1110 bp and encodes a protein of 369 amino acids. The ¢rst nucleotide of the ATG start codon is designated +1 and the deduced amino acid sequence is presented below the open reading frame. Putative regulatory cis elements were found by computer analysis approx. 200 bp upstream from the start codon and included binding sites for AP2 and NGF1A. These recognition sequences have also been identi¢ed by computer analysis in the promotor region of the p35 gene [12]. Also underlined are CA and GA repeats present upstream from the gene. A potential polyadenylation signal is found at positions 2318^2323, approx. 1500 bp downstream from the stop codon. The GenBank accession number for the mouse p39is gene is U90267.
10^53 are, except for a conservative valine to alanine substitution between p39 and p39is, conserved be-
tween the mouse and human proteins and show only limited homology to p35 (Fig. 3). In addition,
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375
Fig. 3. Amino acid sequence alignment of p39, p39is and p35 proteins. Amino acid comparison between the deduced amino acid sequences of human p39 and mouse p39is and mouse p35. The p39 protein consists of 367 amino acids and the p39is polypeptide of 369 residues exhibits 95% identity to the human counterpart. The 307 amino acid long p35 protein displays 57% identity to the human p39is sequence. Shaded areas indicate conserved amino acids between the three proteins. The two underlined sequences show regions of the p35 proteins implied to correspond to the binding and activating domain for CDK5, respectively.
amino acids 86^173 seem to be unique for the p39is/ p39 activator variant (Fig. 3). Possibly, CDK5/p35 and CDK5/p39is exhibit di¡erent substrate preferences. The CDK5/p35 complex has been shown to promote neurite extension of cultured cortical neurons, which agrees with the proposed role of neuronal CDK5 activity in the control of cytoskeletal functions [16]. A targeted disruption of the gene for CDK5 results in abnormal brain development and perinatal death, data which show the importance of CDK5 during brain development [17]. Also, mice
lacking p35 display cortical lamination defects, seizures and adult lethality [18]. Whether a targeted disruption of the p39is gene also results in developmental defects remains to be seen. Thus, several studies have reported the importance of this novel family of kinase complexes during nervous system development. Future studies will provide answers regarding substrate speci¢city and functional roles for this family of kinases in the mature nervous system. This work was supported by the Swedish Natural Research Council, Grants B-AA/BU 09714-311, Axel
BBAEXP 91143 1-7-98
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F. Nilde¨n et al. / Biochimica et Biophysica Acta 1398 (1998) 371^376
References
Fig. 4. Tissue distribution of p39is mRNA. Northern blot showing that the mRNA expression of p39is is neural speci¢c. 20 Wg of total RNA was loaded in each lane. (A) Hybridisation with the p39is speci¢c probe shows high expression of p39is in total brain and cerebellum and moderate levels in the hippocampus and spinal cord. None of the peripheral tissues examined displayed any evident p39is mRNA expression. The size of the transcript was estimated to be approx. 2.6 kb, compared to 18S and 28S ribosomal RNA visualised by ethidium bromide staining. (B) Hybridisation with a GADPH fragment was performed to verify proper transfer to the membrane and to ensure that the level of total RNA loaded/slot were comparable.
and Signe Lagermans Foundation, Pharmacia Research Foundation and the Alzheimer Foundation Sweden (to C.B.). We are grateful to Helena Vretman for skilful technical assistance in DNA sequencing and we thank Dr. Alexander Mercer for helpful revision of the manuscript.
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BBAEXP 91143 1-7-98