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• W2067132930 abstract "The high molecular weight neurofilament protein (NF-H) is highly phosphorylated in the axon. The phosphorylation sites have been identified as KSP (Lys-Ser-Pro) repeats in the tail domain of NF-H. These KSP sequences are present more than 50 times in the NF-H tail, and most of these sites are normally phosphorylated in vivo. These KSP sites can be further divided into two separate consensus sequences, KSPXK and KSPXY (where Y is not K). The extensive phosphorylation of NF-H has been proposed to play a critical role in the determination of axonal diameter. Recent studies have shown that Cdk5, a kinase related to the cell cycle-dependent kinase Cdc2, is expressed in the brain and associates with the cytoskeleton. In vitro phosphorylation studies have shown that Cdk5 in conjunction with its activator, p35, is able to phosphorylate histone H1, dephosphorylated NF-H, as well as a synthetic peptide with the repetitive KSP motif. We have cloned the cDNAs for rat Cdk5 and p35 by reverse transcription-polymerase chain reaction and cDNA library screening and studied the phosphorylation of NF-H both in vivo and in vitro. By transient transfection assays, we have shown that NF-H can only be extensively phosphorylated in the presence of both Cdk5 and p35. This phosphorylation can be inhibited by a Cdk5-dominant negative mutant, an observation which further supports that Cdk5 is a kinase that is able to phosphorylate NF-H. By immunoprecipitating Cdk5 and p35 from the transfected cells, we have been able to show that the KSPXK repeats are the preferred phosphorylation sites for Cdk5, while the KSPXY repeats are not directly phosphorylated by Cdk5 and p35. The high molecular weight neurofilament protein (NF-H) is highly phosphorylated in the axon. The phosphorylation sites have been identified as KSP (Lys-Ser-Pro) repeats in the tail domain of NF-H. These KSP sequences are present more than 50 times in the NF-H tail, and most of these sites are normally phosphorylated in vivo. These KSP sites can be further divided into two separate consensus sequences, KSPXK and KSPXY (where Y is not K). The extensive phosphorylation of NF-H has been proposed to play a critical role in the determination of axonal diameter. Recent studies have shown that Cdk5, a kinase related to the cell cycle-dependent kinase Cdc2, is expressed in the brain and associates with the cytoskeleton. In vitro phosphorylation studies have shown that Cdk5 in conjunction with its activator, p35, is able to phosphorylate histone H1, dephosphorylated NF-H, as well as a synthetic peptide with the repetitive KSP motif. We have cloned the cDNAs for rat Cdk5 and p35 by reverse transcription-polymerase chain reaction and cDNA library screening and studied the phosphorylation of NF-H both in vivo and in vitro. By transient transfection assays, we have shown that NF-H can only be extensively phosphorylated in the presence of both Cdk5 and p35. This phosphorylation can be inhibited by a Cdk5-dominant negative mutant, an observation which further supports that Cdk5 is a kinase that is able to phosphorylate NF-H. By immunoprecipitating Cdk5 and p35 from the transfected cells, we have been able to show that the KSPXK repeats are the preferred phosphorylation sites for Cdk5, while the KSPXY repeats are not directly phosphorylated by Cdk5 and p35. Neurofilaments are 8–10 nm fibrous structures present in all vertebrate axons. In mammals, neurofilaments consist of three protein subunits, known as high (NF-H), 1The abbreviations used are: NF-H, NF-M, and NF-Lneurofilament high, middle, and low molecular weight proteins, respectivelyPAGEpolyacrylamide gel electrophoresisCdkcyclin dependent kinasePCRpolymerase chain reactionRTreverse transcriptasekbkilobase(s)ECLenhanced chemiluminescenceGSK3βglycogen synthase kinase 3βHAhemagglutinin. middle (NF-M), and low (NF-L) molecular weight neurofilament proteins (1Fliegner K.H. Liem R.K.H. Int. Rev. Cytol. 1991; 131: 109-167Google Scholar, 2Liem R.K.H. Curr. Opin. Cell Biol. 1993; 5: 12-16Google Scholar, 3Lee M.K. Cleveland D.W. Curr. Opin. Cell Biol. 1994; 6: 34-40Google Scholar, 4Nixon R.A. Sihag R.K. Trends Neurosci. 1991; 14: 501-506Google Scholar). These three proteins belong to the intermediate filament protein family and are classified along with α-internexin as type IV intermediate filaments. Like all other intermediate filament proteins, neurofilament proteins contain an amino-terminal head domain, a central α-helical domain of ∼310 amino acids, and a carboxyl-terminal tail domain of variable length. The tail domains increase in length with increasing size of the neurofilament proteins and are extensively phosphorylated in NF-M and NF-H (5Carden M.J. Schlaepfer W.W. Lee V.M.-Y. J. Biol. Chem. 1985; 260: 9805-9817Google Scholar, 6Julien J.P. Mushynski W.E. J. Biol. Chem. 1983; 258: 4019-4025Google Scholar). The consensus sequence for phosphorylation has been identified as Lys-Ser-Pro (KSP) (7Lee V.M.-Y. Otvos Jr., L. Carden M.J. Hollosi M. Dietzschold B. Lazzarini R.A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1998-2002Google Scholar, 8Geisler N. Vandekerckhove J. Weber K. FEBS Lett. 1987; 221: 403-407Google Scholar, 9Elhanany E. Jaffe H. Link W.T. Sheeley D.M. Gainer H. Pant H.C. J. Neurochem. 1994; 63: 2324-2335Google Scholar), which can be further divided into two separate consensus sequences, KSPXK and KSPXY (where Y is not K). There are about 50 potential phosphorylation sites in the rat, mouse, and human NF-H carboxyl-terminal tail domains (10Chin S.S.M. Liem R.K.H. J. Neurosci. 1990; 10: 3714-3726Google Scholar), although the relative numbers of KSPXK and KSPXY repeats differ markedly among different species. Phosphate determinations have shown that most of these sites are phosphorylated in vivo (8Geisler N. Vandekerckhove J. Weber K. FEBS Lett. 1987; 221: 403-407Google Scholar, 11Julien J.P. Mushynski W.E. J. Biol. Chem. 1982; 257: 10467-10470Google Scholar, 12Wong J. Hutchison S.B. Liem R.K.H. J. Biol. Chem. 1984; 259: 10867-10874Google Scholar). Rat NF-M contains only 5 KSP sites (13Napolitano E.W. Chin S.S. Colman D.R. Liem R.K.H. J. Neurosci. 1987; 7: 2590-2599Google Scholar), and human NF-M has 12 KSP sites (14Myers M.W. Lazzarini R.A. Lee V.M. Schlaepfer W.W. Nelson D.L. EMBO J. 1987; 6: 1617-1626Google Scholar). Antibodies have been raised which can distinguish phosphorylated and nonphosphorylated KSP epitopes (15Sternberger L.A. Sternberger N.H. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 6126-6130Google Scholar). Several studies have shown that after extensive dephosphorylation by alkaline phosphatase, NF-H migrates more rapidly on SDS-PAGE (5Carden M.J. Schlaepfer W.W. Lee V.M.-Y. J. Biol. Chem. 1985; 260: 9805-9817Google Scholar, 16Lee V.M.-Y. Carden M.J. Schlaepfer W.W. Trojanowski J.Q. J. Neurosci. 1987; 7: 3474-3488Google Scholar). This characteristic mobility shift upon phosphorylation of NF-H and the antibodies specific for the phosphorylated and nonphosphorylated KSP epitopes are therefore useful tools for studying its phosphorylation. neurofilament high, middle, and low molecular weight proteins, respectively polyacrylamide gel electrophoresis cyclin dependent kinase polymerase chain reaction reverse transcriptase kilobase(s) enhanced chemiluminescence glycogen synthase kinase 3β hemagglutinin. The functional role of neurofilament phosphorylation is still not completely clear. Early studies indicated that NF-H might function as a cross-linking protein whose phosphorylation was thought to be important for the formation of cross-linking bridges between neurofilaments (17Hirokawa N. Glicksman M.A. Willard M.B. J. Cell Biol. 1984; 98: 1523-1536Google Scholar, 18Liem R.K. Pachter J.S. Napolitano E.W. Chin S.S. Moraru E. Heimann R. Ann N. Y. Acad Sci. 1985; 455: 492-508Google Scholar). However, a later study showed that nonphosphorylated NF-H was also situated between intermediate filaments (19Hisanaga S. Hirokawa N. J. Neurosci. 1989; 9: 959-966Google Scholar). More recently it has been suggested that the phosphate groups of NF-H may result in electrostatic repulsion, which in turn could increase axonal diameter (20Shaw G. The Neuronal Cytoskeleton. Alan R. Liss, New York1991: 185Google Scholar, 21Carden M.J. Trojanowski J.Q. Schlaepfer W.W. Lee V.M.-Y. J. Neurosci. 1987; 7: 3489-3504Google Scholar). Phosphorylation of NF-H has also been correlated with its ability to interact with microtubules (22Hisanaga S. Kusubata M. Okumura E. Kishimoto T. J. Biol. Chem. 1991; 266: 21798-21803Google Scholar). The kinase(s) which phosphorylate the KSP sequences on NF-H are still not completely identified. A number of different protein kinases, including casein kinase (23Floyd C.C. Grant P. Gallant P.E. Pant H.C. J. Biol. Chem. 1991; 266: 4987-4994Google Scholar), cyclic AMP-dependent protein kinase (24Dosemeci A. Pant H.C. Biochem. J. 1992; 282: 477-481Google Scholar), Ca2+-calmodulin-dependent protein kinase (25Vallano M.L. Buckholz T.M. DeLorenzo R.J. Biochem. Biophys. Res. Commun. 1985; 130: 957-963Google Scholar), and protein kinase C (26Sihag R.K. Nixon R.A. J. Biol. Chem. 1990; 265: 4166-4171Google Scholar) are able to phosphorylate the neurofilament proteins. However, NF-M is a better substrate than NF-H for all these protein kinases, and the characteristic mobility shift on SDS-PAGE due to phosphorylation of NF-H is not observed. Other studies have revealed protein kinases which are associated with neurofilaments, including a protein kinase activity from bovine spinal cord neurofilament-enriched preparations (27Dosemeci A. Floyd C.C. Pant H.C. Cell Mol. Neurobiol. 1990; 10: 369-382Google Scholar) and a 115-kDa neurofilament-associated kinase isolated by affinity chromatography using bacterially produced NF-H (28Xiao J. Monteiro M.J. J. Neurosci. 1994; 14: 820-833Google Scholar). These protein kinases also only phosphorylate NF-H in a limited manner and there is no evidence that they phosphorylate NF-H at the KSP sites. Recent results have pointed to the family of cyclin-dependent protein kinases (Cdks) as candidate kinases for the KSP sites on NF-H. The cell cycle kinase Cdc2 was shown to phosphorylate neurofilaments (29Guan R.J. Hall F.L. Cohlberg J.A. J. Neurochem. 1992; 58: 1365-1371Google Scholar, 30Hisanaga S. Kusubata M. Okumura E. Kishimoto T. J. Biol. Chem. 1991; 266: 21798-21803Google Scholar) and cause the characteristic gel mobility shift of NF-H upon phosphorylation (30Hisanaga S. Kusubata M. Okumura E. Kishimoto T. J. Biol. Chem. 1991; 266: 21798-21803Google Scholar). However, these results do not have much physiological relevance, since it is known that Cdc2 kinase is absent in terminally differentiated neurons. By using the polymerase chain reaction (PCR) with degenerate oligonucleotide primers corresponding to conserved regions of the Cdks, a family of novel Cdc2-related cDNA clones have been isolated (31Meyerson M. Enders G.H. Wu C.L. Su L.K. Gorka C. Nelson C. Harlow E. Tsai L.H. EMBO J. 1992; 11: 2909-2917Google Scholar). Among them, PSSALRE is expressed in neuronal cell lines and brain, as well as other cell lines and organs (31Meyerson M. Enders G.H. Wu C.L. Su L.K. Gorka C. Nelson C. Harlow E. Tsai L.H. EMBO J. 1992; 11: 2909-2917Google Scholar). This same kinase was also separately identified by another laboratory, shown to be able to bind cyclin D1 (32Xiong Y. Zhang H. Beach D. Cell. 1992; 71: 505-514Google Scholar) and renamed as Cdk5 (cyclin-dependent kinase 5). A number of recent studies have shown that Cdk5 is associated with the cytoskeleton and able to phosphorylate NF-H in vitro. (33Lew J. Winkfein R.J. Paudel H.K. Wang J.H. J. Biol. Chem. 1992; 267: 25922-25926Google Scholar, 34Shetty K.T. Link W.T. Jaffe H. Wang J. Pant H.C. J. Neurochem. 1995; 64: 2681-2690Google Scholar, 35Hisanaga S. Ishiguro K. Uchida T. Okumura E. Okano T. Kishimoto T. J. Biol. Chem. 1993; 268: 15056-15060Google Scholar). An additional protein with a molecular mass of 23–25 kDa was observed in most of the Cdk5 preparations. cDNA cloning showed that this 23–25-kDa protein is a degradation product of a larger protein, p35, which acts as a neural-specific regulatory subunit of Cdk5 (36Tsai L.H. Delalle I. Caviness Jr., V.S. Chae T. Harlow E. Nature. 1994; 371: 419-423Google Scholar, 37Lew J. Huang Q.Q. Zhong Q. Winkfein R.J. Aebersold R. Hunt T. Wang J.H. Nature. 1994; 371: 423-426Google Scholar). p35 associates physically with and activates Cdk5 kinase. Although it has no homology with cyclins, it serves a similar function in modulating the activity of Cdk5. In this study, we have cloned the cDNAs for both Cdk5 and p35 from rat brain and conducted NF-H phosphorylation studies by transient transfections and immunoprecipitation/kinase assays. We have demonstrated that NF-H can be phosphorylated only in the presence of both Cdk5 and p35. Furthermore, this phosphorylation is specific for the KSPXK sites. Rat Cdk5 cDNA was amplified from mRNA by the RT-PCR method. Briefly, 1 µg of poly(A)+ RNA from rat brain (Clontech) was primed with random hexamers and reverse-transcribed with Moloney murine leukemia virus reverse transcriptase (RT) according to the manufacturer's protocol (Perkin-Elmer). For amplification of Cdk5 cDNA, the RT reaction was added to a PCR reaction mixture, containing reaction buffer, 2.5 units of Taq DNA polymerase, sense (5′-CGAAGCTTGGACTCTTAGAACCGA-3′) and antisense (5′-TGGAAGCTTGGCTTAAATAGGTCAGG-3′) oligonucleotide primers (Operon) corresponding to the published rat Cdk5 sequence. This reaction was heated to 94°C for 5 min, cooled to 72°C for 5 min, and cycled 30 times at 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min. The resulting rat Cdk5 PCR product was subcloned into the pGEM-7 (Promega) and pRSVi vectors and sequenced in its entirety from both directions. Bovine p23 cDNA was obtained by the same approach with sense (5′-GGCATATGTCGTCCGTCAAGAAGG-3′) and antisense (5′-ATGCATATGGCTGGCGGGCTCACC-3′) primers corresponding to the published sequence of bovine p23 (34Shetty K.T. Link W.T. Jaffe H. Wang J. Pant H.C. J. Neurochem. 1995; 64: 2681-2690Google Scholar). The bovine p23 PCR product was subcloned into pGEM-5 and pRSVi vectors and used in screening a rat brain λgt11 cDNA library (generously provided by Dr. David Colman at Mt. Sinai Medical Center). Standard cDNA library screening procedures were employed (38Sambrook J. Fritsh E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Briefly, the 0.5-kb HindIII fragment containing the bovine p23 cDNA was purified from pRSVi-p23, labeled with 32P by random primer synthesis (Amersham) and used as a probe for screening the amplified λgt11 rat brain cDNA expression library (13Napolitano E.W. Chin S.S. Colman D.R. Liem R.K.H. J. Neurosci. 1987; 7: 2590-2599Google Scholar). After screening approximately 1 × 105 plaques, 20 positive plaques were recovered and confirmed to correspond to be rat p35 by secondary and tertiary screening. The largest p35 cDNA clone was isolated from the phage, subcloned into the EcoRI site of pGEM-7 (Promega), and sequenced in its entirety from both directions. The Cdk5 dominant negative construct was generated by two-step PCR (39Barik S. Methods Mol. Biol. 1993; 15: 277-286Google Scholar). The first amplification was performed using an antisense primer corresponding to the amino acid residues KLAD*FGL (where * marks the point of a mutation, which contains a single G to A mismatch resulting in a Asp → Asn mutation (40van den Heuvel S. Harlow E. Science. 1993; 262: 2050-2054Google Scholar)) and a sense primer corresponding to the 5′ end of Cdk5 described above. The resulting PCR product was purified and used with the Cdk5 antisense primer described above for the second amplification. The final PCR product was subcloned into the pRSVi vector and sequenced to confirm the Asp → Asn mutation. The 3.4-kb cDNA of rat NF-H encoding the full-length polypeptide was previously cloned into the pRSVi-HindIII vector (10Chin S.S.M. Liem R.K.H. J. Neurosci. 1990; 10: 3714-3726Google Scholar), which contains the Rous sarcoma virus long terminal repeat (41Forman B.M. Yang C.R. Stanley F. Casanova J. Samuels H.H. Mol. Endocrinol. 1988; 2: 902-911Google Scholar). The full-length NF-H cDNA was also cloned into the expression vector pET-16b (Novagen Inc.). The truncated NF-H tail constructs which contain either the KSPXK or KSPXY sequences were generated by PCR. Two pairs of primers (Operon) were designed according to the NF-H sequence (10Chin S.S.M. Liem R.K.H. J. Neurosci. 1990; 10: 3714-3726Google Scholar). Primers KSP1 (5′-GAACATATGTCTCCTGTGAAAGAA-3′) and KSP2 (5′-TCTGGATCCTTTATGGAGATTTTA-3′) were for the KSPXY-containing construct, whereas primers KSP3 (5′-GCCCATATGCCCGTGAAGGAAGGT-3′) and KSP4 (5′-GAGGGATCCGGGCTATTCTGGGTG-3′) were for the KSPXK-containing construct. PCR was carried out as described above, and the resulting PCR products were subcloned in pET16b at the NdeI and BamHI sites and sequenced. GSK3β cDNA was cloned using sense (5′-AGGTCATATGAGAAGAGCCATC-3′) and antisense (5′-AAACCATCTGAGTGTTGCTGAGTG-3′) primers based on the published sequence (61Woodgett J.R. EMBO J. 1990; 9: 2431-2438Google Scholar) by RT-PCR. The resulting PCR product was cloned into pGEM-5 and pRSVi vectors and sequenced in its entirety from both directions. The SW13.cl.2Vim− cell line was the kind gift of Dr. Robert M. Evans (University of Colorado Health Science Center, Denver, CO) and was grown in Dulbecco's modified Eagle's/Ham's F12 medium (Life Technologies, Inc.) supplemented with 5% fetal bovine serum at 37°C in a humidified atmosphere of 5% CO2. All the transient transfections in this study were done using the calcium phosphate precipitation procedure previously described (44Ching G.Y. Liem R.K.H. J. Cell Biol. 1993; 122: 1323-1335Google Scholar). The transiently transfected cells were lysed in 6.25 mM Tris buffer, pH 6.5 and 1% SDS, an equal amount of sample buffer was added, and the samples were boiled for 10 min. SDS-PAGE was performed by the method of Laemmli (43Laemmli U.K. Nature. 1970; 227: 680-685Google Scholar) in different percentage vertical slab gels. For immunoblots, the proteins were separated by SDS-PAGE, transferred to nitrocellulose (Schleicher and Schuell) and probed with the different antibodies. ECL (Amersham) was performed according to the manufacturer's protocols, and the blots were exposed to x-ray film (Kodak). Antibodies SMI36 and SMI32 were purchased from Sternberger Monoclonals Inc. The Cdk5 antibodies C-8 and DC-17 were purchased from Santa Cruz Biotech. The pET constructs encoding NF-H, “KSPXY” and “KSPXK” proteins were transformed into the Escherichia coli strain BL21(DE3). The transformed bacteria were cultured in 500 ml of LB containing 100 µg/ml ampicillin to A600 of 0.5 at 37°C. After adding isopropyl-1-thio-β-D-galactopyanoside to a final concentration of 0.4 mM, the cultures were incubated another 4 h. The bacteria were harvested by centrifugation at 2000 × g and the “KSPXY” and “KSPXK” proteins, which contained the histidine tag from pET16b were further purified according to the manufacturer's protocol (Novagen). Briefly, the bacterial pellets were sonicated in binding buffer containing 5 mM imidazole, 0.5 M NaCl, and 20 mM Tris-HCl, pH 7.9, and centrifuged at 20,000 × g to collect the inclusion bodies and cellular debris, while leaving other proteins in solution. Repeating the previous step several times released more of the trapped soluble proteins. The final pellets were sonicated in binding buffer prepared in 6 M urea and incubated on ice for 1 h. The remaining insoluble material was removed by centrifugation at 39,000 × g, and the supernatants were filtered through 0.45-micron membranes. For the “KSPXK” and “KSPXY” proteins, the filtered supernatants were loaded onto NiSO4 columns, equilibrated according to the manufacturer's protocol, washed with binding buffer, and eluted with elution buffer. All the buffers used in chromatography were prepared in 6 M urea. The eluted fractions were dialyzed overnight against phosphate-buffered saline at 4°C, and the protein concentrations were determined by the Bradford assay (Bio-Rad). The NiSO4 column was not effective for the purification of full-length NF-H, and we therefore purified full-length NF-H using an HTP column as described elsewhere (42Kaplan M.P. Chin S.S. Macioce P. Srinawasan J. Hashim G. Liem R.K.H. J. Neurosci. Res. 1991; 30: 45-54Google Scholar); the protein concentrations were measured by the same method. Transiently transfected cells were washed with phosphate-buffered saline and incubated in lysis buffer containing 50 mM Tris-HCl, pH 7.5, 250 mM NaCl, 5 mM EDTA, 5 mM dithiothreitol, 0.1% Nonidet P-40 (Sigma), and 1 × proteinase inhibitors (5 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin) for 10 min at 4°C. Cells were then scraped into Eppendorf tubes and incubated on ice for 1 h. The cell suspensions were pelleted by centrifugation at 14,000 × g for 10 min, and the supernatants were mixed with 1 µg of antibody for 2 h at 4°C. 20 µl of Protein G Plus-Agarose (Santa Cruz Biotech) was added, and the mixtures were subsequently incubated for 2 h at 4°C. The immune complexes were pelleted by centrifugation at 14,000 × g and washed with cold lysis buffer four times. The kinase assays were initiated by mixing kinase buffer containing 50 mM Hepes, pH 7.4, 5 mM MgCl2, 5 mM MnCl2, 1 mM dithiothreitol, 1 mM EDTA, 100 mM NaCl, 1 µCi of [γ-32P]ATP (Amersham), and 2 mg of substrate with the final immune complex pellet. The reaction mixtures were then incubated in a 30°C water bath for 30 min and centrifuged at 14,000 × g for 10 min. The supernatants were saved and the pellets were washed 3 times. An equal amount of 2 × sample buffer (43Laemmli U.K. Nature. 1970; 227: 680-685Google Scholar) were added to the supernatants and pellets, and the samples were boiled for 10 min before SDS-PAGE. In order to study Cdk5 and its activator, p35, we first isolated their cDNAs by RT-PCR. To obtain the Cdk5 cDNA, reverse transcription was carried out using rat brain mRNA as template, and the resulting RT-PCR product was amplified by two primers complementary to the 5′ and 3′ ends of the published Cdk5 cDNA sequence (45Hellmich M.R. Pant H.C. Wada E. Battey J.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10867-10871Google Scholar). This PCR product was subcloned into the pGEM-5 vector, its sequence was determined and confirmed to be identical with the Cdk5 sequence from GenBank™. The full-length rat p35 cDNA was obtained by first isolating a cDNA corresponding to the partial sequence of bovine p35 (p23) by RT-PCR using bovine brain mRNA as template (46Ishiguro K. Kobayashi S. Omori A. Takamatsu M. Yonekura S. Anzai K. Imahori K. Uchida T. FEBS Lett. 1994; 342: 203-208Google Scholar) and screening a rat brain cDNA library using the bovine p23 cDNA as a probe. The resulting p35 cDNA was subcloned into pGEM-5 and sequenced. The deduced amino acid sequence of rat p35 is nearly identical with those of human and bovine p35 (Fig. 1). There are only four amino acid differences between rat and human p35 (36Tsai L.H. Delalle I. Caviness Jr., V.S. Chae T. Harlow E. Nature. 1994; 371: 419-423Google Scholar) or between rat and bovine p35 (37Lew J. Huang Q.Q. Zhong Q. Winkfein R.J. Aebersold R. Hunt T. Wang J.H. Nature. 1994; 371: 423-426Google Scholar). The Cdk5 dominant-negative (Cdk5dn) construct was generated by PCR and subcloned into the expression vectors described under “Materials and Methods.” This dominant-negative Cdk5dn contains a point mutation resulting in a single amino acid residue change (Asp144→ Asn) in the kinase domain of Cdk5. This mutant form has previously been shown to result in an inactive kinase, which competes with the activator protein p35 and thereby inhibits Cdk5 kinase activity (40van den Heuvel S. Harlow E. Science. 1993; 262: 2050-2054Google Scholar). A full-length cDNA for glycogen synthase kinase 3β (GSK3β) was also generated by RT-PCR using primers based on the published sequence (61Woodgett J.R. EMBO J. 1990; 9: 2431-2438Google Scholar). The resulting GSK3β cDNA was subcloned into pGEM-5 and sequenced. The sequence was found to be identical with the GSK3β sequence obtained from GenBank™. Rat NF-H contains 52 KSP repeats, which can be divided into KSPXK and KSPXY repeats. After analyzing the protein sequence, we noticed that most of the KSPXY repeats are clustered in the amino-terminal part of the NF-H tail domain, while most of the KSPXK repeats are located after the KSPXY repeats. Using PCR, we were able to separate these 2 groups of repeats and prepare a “KSPXY” construct which contains 41 KSPXY repeats and no KSPXK repeats and a “KSPXK” construct which contains 7 KSPXK repeats (and 2 KSPXY repeats). These constructs were subcloned into the pET16b expression vector (Fig. 2). It should be noted that a single KSPXK is present in front of the cluster of the KSPXY repeats in the NF-H sequence and the “KSPXY” construct starts from the middle of this KSPXK motif. To study the phosphorylation of NF-H by Cdk5, transient transfections were performed with SW13cl2Vim− cells, a human adrenal carcinoma cell line without cytoplasmic intermediate filaments (47Sattia A.J. Nordeen S.K. Evans R.M. J. Cell Biol. 1990; 111: 553-565Google Scholar). We have previously shown by Western blot analysis and immunostaining that NF-H is predominantly in the nonphosphorylated form in transiently transfected fibroblasts (10Chin S.S.M. Liem R.K.H. J. Neurosci. 1990; 10: 3714-3726Google Scholar). The same result is obtained from SW13cl2Vim− cells transiently transfected with NF-H alone (Fig. 3B). Cdk5 is expressed in a number of cell lines and tissues, but its activity has been shown to be present only in brain (48Tsai L.H. Takahashi T. Caviness Jr., V.S. Harlow E. Development. 1993; 119: 1029-1040Google Scholar). Consistent with this observation, Cdk5 protein can be detected in SW13cl2Vim− cells by Western blot analysis (Fig. 3C), but the immunoprecipitated Cdk5 from cells lacking p35 does not have kinase activity on histone H1 (Fig. 4A), NF-H (Fig. 4B), “KSPXK” (Fig. 4C), or “KSPXY” proteins (Fig. 4D). Several recent reports have shown that p35 is only expressed in the central nervous system (36Tsai L.H. Delalle I. Caviness Jr., V.S. Chae T. Harlow E. Nature. 1994; 371: 419-423Google Scholar, 37Lew J. Huang Q.Q. Zhong Q. Winkfein R.J. Aebersold R. Hunt T. Wang J.H. Nature. 1994; 371: 423-426Google Scholar). Consistent with these reports, our Northern blot analysis showed that the SW13cl2Vim− cell line does not have any p35 mRNA (data not shown). These results indicate that the SW13cl2Vim− cell line is a good system to study the phosphorylation of NF-H by Cdk5 and its activator, p35.Fig. 4In vitro kinase assays. Extracts from cells, which had been transiently transfected with combinations of Cdk5, p35, and Cdk5dn constructs were immunoprecipitated with the Cdk5 antibody C-8 and used for the in vitro kinase assays. Histone H1 was used as a positive control (A). Bacterially expressed NF-H (B), “KSPXK” (C), and “KSPXY” (D) proteins were purified and used as substrates for the in vitro kinase assays. Lane 7 shows the Coomassie Blue stain of the substrates.View Large Image Figure ViewerDownload (PPT) For every transient transfection experiment, 20 µg of each plasmid was used, and the resulting cell lysates were analyzed by Western blots using antibodies SMI36 and SMI32. SMI36 is specific for the phosphorylated KSP epitope on NF-H, while SMI32 recognizes this epitope in the nonphosphorylated form (15Sternberger L.A. Sternberger N.H. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 6126-6130Google Scholar). Purified NF-H from rat spinal cord shows an apparent molecular mass of approximately 200 kDa on SDS-PAGE. After alkaline phosphatase treatment, the dephosphorylated NF-H migrates faster on SDS-PAGE (5Carden M.J. Schlaepfer W.W. Lee V.M.-Y. J. Biol. Chem. 1985; 260: 9805-9817Google Scholar, 49Lee V.M.-Y. Carden M.J. Schlaepfer W.W. Trojanowski J.Q. J. Neurosci. 1987; 7: 3474-3488Google Scholar). We used this mobility shift on SDS-PAGE and the immunoreactivity of NF-H with the two phosphorylation state-dependent antibodies as the criteria to determine whether or not the transfected NF-H was phosphorylated. As shown in Fig. 3, when NF-H is transfected by itself (lane 2) or co-transfected with Cdk5 (lane 3) into SW13cl2Vim− cells, the exogenously expressed NF-H protein is detected only by SMI32 and does not show the mobility shift characteristic of the phosphorylated NF-H, indicating the absence of any significant amount of NF-H phosphorylation (Fig. 3, A and B). However, when both Cdk5 and p35 are transfected along with NF-H (Fig. 3, lane 6), the expressed NF-H shows an apparent molecular mass of 200 kDa, comparable to that of the phosphorylated NF-H. Furthermore, the protein is recognized by SMI36 (Fig. 3A), which is specific for the phosphorylated NF-H, but not by SMI32 (Fig. 3B), which detects the nonphosphorylated KSP sequences on NF-H. From these results, it is quite clear that the NF-H expressed in the transfected cells is extensively phosphorylated. When NF-H is co-transfected with only p35 (Fig. 3, lane 5), a slight mobility shift of the NF-H band is observed on the immunoblots and this broadly diffuse band is recognized by both SMI36 and SMI32 (Fig. 3, A and B). These results are consistent with partial phosphorylation of NF-H by the endogenous Cdk5 activated by the transfected p35. However, the endogenous Cdk5 is apparently not present in sufficient quantity to phosphorylate NF-H to the same degree as the exogenous Cdk5 (Fig. 3, A and B, lanes 5 and 6). When the dominant-negative Cdk5dn is introd" @default.
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• W2067132930 title "Phosphorylation of the High Molecular Weight Neurofilament Protein (NF-H) by Cdk5 and p35" @default.
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