Functional Consequences of Mutations in CDKL5, an X-linked Gene Involved in Infantile Spasms and Mental Retardation

Abstract

Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been identified in patients with Rett syndrome, West syndrome, and X-linked infantile spasms sharing the common features of generally intractable early seizures and mental retardation. Disease-causing mutations are distributed in both the catalytic domain and in the large COOH terminus. In this report, we examine the functional consequences of some Rett mutations of CDKL5 together with some synthetically designed derivatives useful to underline the functional domains of the protein. The mutated CDKL5 derivatives have been subjected to in vitro kinase assays and analyzed for phosphorylation of the TEY (Thr-Glu-Tyr) motif within the activation loop, their subcellular localization, and the capacity of CDKL5 to interact with itself. Whereas wild-type CDKL5 autophosphorylates and mediates the phosphorylation of the methyl-CpG-binding protein 2 (MeCP2) in vitro, Rett-mutated proteins show both impaired and increased catalytic activity suggesting that a tight regulation of CDKL5 is required for correct brain functions. Furthermore, we show that CDKL5 can self-associate and mediate the phosphorylation of its own TEY (Thr-Glu-Tyr) motif. Eventually, we show that the COOH terminus regulates CDKL5 properties; in particular, it negatively influences the catalytic activity and is required for its proper sub-nuclear localization. We propose a model in which CDKL5 phosphorylation is required for its entrance into the nucleus whereas a portion of the COOH-terminal domain is responsible for a stable residency in this cellular compartment probably through protein-protein interactions. Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been identified in patients with Rett syndrome, West syndrome, and X-linked infantile spasms sharing the common features of generally intractable early seizures and mental retardation. Disease-causing mutations are distributed in both the catalytic domain and in the large COOH terminus. In this report, we examine the functional consequences of some Rett mutations of CDKL5 together with some synthetically designed derivatives useful to underline the functional domains of the protein. The mutated CDKL5 derivatives have been subjected to in vitro kinase assays and analyzed for phosphorylation of the TEY (Thr-Glu-Tyr) motif within the activation loop, their subcellular localization, and the capacity of CDKL5 to interact with itself. Whereas wild-type CDKL5 autophosphorylates and mediates the phosphorylation of the methyl-CpG-binding protein 2 (MeCP2) in vitro, Rett-mutated proteins show both impaired and increased catalytic activity suggesting that a tight regulation of CDKL5 is required for correct brain functions. Furthermore, we show that CDKL5 can self-associate and mediate the phosphorylation of its own TEY (Thr-Glu-Tyr) motif. Eventually, we show that the COOH terminus regulates CDKL5 properties; in particular, it negatively influences the catalytic activity and is required for its proper sub-nuclear localization. We propose a model in which CDKL5 phosphorylation is required for its entrance into the nucleus whereas a portion of the COOH-terminal domain is responsible for a stable residency in this cellular compartment probably through protein-protein interactions. X-linked cyclin-dependent kinase-like 5 (CDKL5, 3The abbreviations used are: CDKL5, cyclin-dependent kinase-like 5; MAP, mitogen-activated protein; MAPK, mitogen-activated protein kinase; MeCP2, methyl-CpG-binding protein 2; GFP, green fluorescent protein; ERK, extracellular signal-regulated kinase. 3The abbreviations used are: CDKL5, cyclin-dependent kinase-like 5; MAP, mitogen-activated protein; MAPK, mitogen-activated protein kinase; MeCP2, methyl-CpG-binding protein 2; GFP, green fluorescent protein; ERK, extracellular signal-regulated kinase. previously named STK9) was originally identified in a transcriptional mapping project focused on the human chromosome Xp22.3-p21.3, spanning a region critical for several diseases. Expression studies demonstrated that CDKL5 was transcribed in several tissues, including brain (1Montini E. Andolfi G. Caruso A. Buchner G. Walpole S.M. Mariani M. Consalez G. Trump D. Ballabio A. Franco B. Genomics. 1998; 51: 427-433Crossref PubMed Scopus (110) Google Scholar). However, the possible link between CDKL5 and human diseases was drawn only few years later when balanced translocating events disrupting the gene were identified in two female patients affected by severe infantile spasms and mental retardation (2Kalscheuer V.M. Tao J. Donnelly A. Hollway G. Schwinger E. Kubart S. Menzel C. Hoeltzenbein M. Tommerup N. Eyre H. Harbord M. Haan E. Sutherland G.R. Ropers H.H. Ge´cz J. Am. J. Hum. Genet. 2003; 72: 1401-1411Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). Retrospectively, a previous publication had identified a large deletion involving CDKL5 in a male patient with X-linked retinoschisis and seizure (3Huopaniemi L. Tyynismaa H. Rantala A. Rosenberg T. Alitalo T. Hum. Mutat. 2000; 16: 307-314Crossref PubMed Scopus (39) Google Scholar); it has recently been hypothesized that retinoschisis is due to deletion of the XLRS1 gene, whereas epilepsy is caused by truncation of at least the last exon of CDKL5 (2Kalscheuer V.M. Tao J. Donnelly A. Hollway G. Schwinger E. Kubart S. Menzel C. Hoeltzenbein M. Tommerup N. Eyre H. Harbord M. Haan E. Sutherland G.R. Ropers H.H. Ge´cz J. Am. J. Hum. Genet. 2003; 72: 1401-1411Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). The importance of CDKL5 in early onset seizures and severe mental retardation in females has been further reinforced by five recent reports linking mutations in CDKL5 to patients with Rett syndrome but only in those affected by a variant form characterized by seizure onset before 6 months of age (4Tao J. Van Esch H. Hagedorn-Greiwe M. Hoffmann K. Moser B. Raynaud M. Sperner J. Fryns J.-P. Schwinger E. Ge´cz J. Ropers H.-H. Kalscheuer V.M. Am. J. Hum. Genet. 2004; 75: 1149-1154Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar, 5Weaving L.S. Christodoulou J. Williamson S.L. Friend K.L. McKenzie O.L.D. Archer H. Evans J. Clarke A. Pelka G.J. Tam P.P.L. Watson C. Lahooti H. Ellaway C.J. Bennetts B. Leonard H. Ge´cz J. Am. J. Hum. Genet. 2004; 75: 1079-1093Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar, 6Scala E. Ariani F. Mari F. Caselli R. Pescucci C. Longo I. Meloni I. Giachino D. Bruttini M. Hayek G. Zappella M. Renieri A. J. Med. Genet. 2005; 42: 103-107Crossref PubMed Scopus (209) Google Scholar, 7Evans J.C. Archer H.L. Colley J.P. Ravn K. Nielsen J.B. Kerr A. Williams E. Christodoulou J. Gecz J. Jardine P.E. Wright M.J. Pilz D.T. Lazarou L. Cooper D.N. Sampson J.R. Butler R. Whatley S.D. Clarke A.J. Eur. J. Hum. Genet. 2005; 13: 1113-1120Crossref PubMed Scopus (160) Google Scholar, 8Mari F. Azimonti S. Bertani I. Bolognese F. Colombo E. Caselli R. Scala E. Longo I. Grosso S. Pescucci C. Ariani F. Hayek G. Balestri P. Bergo A. Badaracco G. Zappella M. Broccoli V. Renieri A. Kilstrup-Nielsen C. Landsberger N. Hum. Mol. Genet. 2005; 14: 1935-1946Crossref PubMed Scopus (264) Google Scholar). Very recently the frequency of CDKL5 mutations in patients affected by infantile spasms or early onset epilepsy of unknown cause has been investigated. The identification of several novel likely pathogenic mutations led the authors to propose that CDKL5 mutations in females are a significant cause of severe mental retardation and of forms of early epilepsy that are generally intractable (9Archer H.L. Evans J. Edwards S. Colley J. Newbury-Ecob R. O'Callaghan F. Huyton M. O'Regan M. Tolmie J. Sampson J. Clarke A. Osborne J. J. Med. Genet. 2006; 43: 729-734Crossref PubMed Scopus (164) Google Scholar). Despite the clear involvement of CDKL5 in human health, this protein remains almost completely uncharacterized; presently, we are missing its functions in the nervous system as well as the molecular consequences of its pathogenic mutations. Given its amino acids sequence, CDKL5 was supposed to be a proline-directed serine/threonine kinase sharing homology with members of the mitogen-activated protein (MAP) kinase and cyclin-dependent kinase (CDK) families (1Montini E. Andolfi G. Caruso A. Buchner G. Walpole S.M. Mariani M. Consalez G. Trump D. Ballabio A. Franco B. Genomics. 1998; 51: 427-433Crossref PubMed Scopus (110) Google Scholar). Moreover, the similar Rett phenotypes in patients carrying mutations in the gene coding for the methyl-CpG-binding protein 2 (MECP2) and CDKL5, together with recent reports demonstrating the importance of MeCP2 phosphorylation for selective binding to DNA (10Chen W.G. Chang Q. Lin Y. Meissner A. West A.E. Griffith E.C. Jaenisch R. Greenberg M.E. Science. 2003; 302: 885-889Crossref PubMed Scopus (1011) Google Scholar), opened the possibility that the two proteins might operate in a common developmental pathway. In favor of this hypothesis, we have recently shown that generally the two genes show spatial and temporal overlapping expression pattern that is simultaneously activated by neural maturation and synaptogenesis. Moreover, we demonstrated that CDKL5 and MeCP2 interact and that immunopurified CDKL5 leads, directly or indirectly, to the phosphorylation of the methyl-binding protein (8Mari F. Azimonti S. Bertani I. Bolognese F. Colombo E. Caselli R. Scala E. Longo I. Grosso S. Pescucci C. Ariani F. Hayek G. Balestri P. Bergo A. Badaracco G. Zappella M. Broccoli V. Renieri A. Kilstrup-Nielsen C. Landsberger N. Hum. Mol. Genet. 2005; 14: 1935-1946Crossref PubMed Scopus (264) Google Scholar). The interaction between MeCP2 and CDKL5 has also been confirmed by Lin and colleagues; however, the failure in reproducing the MeCP2 phosphorylation led the authors to propose that MeCP2 might target CDKL5 to DNA-binding complexes containing a different functional substrate of the enzyme (11Lin C. Franco B. Rosner M.R. Hum. Mol. Genet. 2005; 14: 3775-3786Crossref PubMed Scopus (93) Google Scholar). Integrating all data so far available, the emerging picture is that, in fact, CDKL5 and MeCP2 work in common molecular pathways. However, the demonstration that CDKL5 mutations are an important etiological factor for neurodevelopmental disorders in addition to Rett syndrome (9Archer H.L. Evans J. Edwards S. Colley J. Newbury-Ecob R. O'Callaghan F. Huyton M. O'Regan M. Tolmie J. Sampson J. Clarke A. Osborne J. J. Med. Genet. 2006; 43: 729-734Crossref PubMed Scopus (164) Google Scholar), together with our previous expression studies showing that in some specific cerebellar domains the two genes are independently regulated (8Mari F. Azimonti S. Bertani I. Bolognese F. Colombo E. Caselli R. Scala E. Longo I. Grosso S. Pescucci C. Ariani F. Hayek G. Balestri P. Bergo A. Badaracco G. Zappella M. Broccoli V. Renieri A. Kilstrup-Nielsen C. Landsberger N. Hum. Mol. Genet. 2005; 14: 1935-1946Crossref PubMed Scopus (264) Google Scholar), suggest that the kinase might also have functions not connected to the methyl-binding protein. In this study we characterize several CDKL5-mutated derivatives, including some of those identified in Rett patients. Phosphorylation assays performed with the wild-type protein confirm its capability to mediate the modification of MeCP2 in vitro, whereas Rett missense mutations within the conserved catalytic domain abrogate or significantly impair the enzymatic activity. Interestingly, none of these mutants maintains the capability to influence the phosphorylation status of the methyl-binding protein. Frameshift mutations, which generate truncated proteins in patients, have been found both in the amino- and carboxyl-terminal regions of CDKL5. We therefore analyzed two derivatives interrupting the unusually large COOH-terminal region. Our results suggest that the COOH terminus of the protein has regulatory functions that can influence either the catalytic activity or the subcellular localization. We conclude that both a change in CDKL5 activity and its mislocalization can be responsible for the onset of neurodevelopmental disorders and believe that these studies should help in drawing some speculations on the CDKL5 mutation genotype/phenotype correlation. Plasmid Construction—Recombinant hMeCP2E2 was produced from the pTYB1-hMeCP2 vector containing the entire cDNA of MeCP2 (486 amino acids) cloned into the NdeI and XhoI sites of pTYB1 (New England Biolabs) generating a chitin-binding fusion protein. Recombinant hCDKL5 encompassing amino acids 301-751 for immunizing rabbits was produced as a chitin-binding fusion protein from pTYB1-CDKL5301-751. The corresponding cDNA was generated by PCR and cloned into the NdeI and SapI sites of pTYB1. pGFP-CDKL5 contains the entire cDNA of hCDKL5 (1030 amino acids) generated by PCR and cloned into the BspEI-BamHI sites of pEGFP-C1 (Clontech). Subsequently, an extra BamHI site, allowing the excision of the CDKL5 cDNA with BamHI, has been introduced downstream of the BspEI site. The C152F, R175S, and K42R derivatives were generated by site-directed mutagenesis using the QuikChange® site-directed mutagenesis kit (Stratagene). ΔN contains amino acids 298-1030, Δ781 amino acids 1-780, and Δ525 1-524. The analogous FLAG-CDKL5 derivatives (NH2-terminal FLAG-tag) were expressed from the pCMV-Tag-2B vector (Stratagene) into which the cDNAs were cloned into BamHI. All PCR-generated constructs were verified by sequencing. Antibodies—A rabbit anti-CDKL5 anti-serum (Covance Research Products Inc.) was raised against hCDKL5 amino acids 301-751 expressed in Escherichia coli as a fusion protein containing a chitin-binding domain that was eliminated during the purification procedure. For immunopurification and Western blots the following antibodies were used: monoclonal anti-FLAG (Sigma), polyclonal anti-MeCP2 (Sigma), monoclonal anti-GFP (Roche), and polyclonal anti-phospho-p44/42 MAP kinase antibody (Cell Signaling). Recombinant Protein Purification—Recombinant full-length hMeCP2E2 and hCDKL5 amino acids 301-751 were expressed in E. coli ER2566 and induced with 0.5 mm isopropyl β-d-thiogalactopyranoside at 30 °C for 5 h. Following induction, extracts were prepared by resuspending the bacteria in lysis buffer (750 mm NaCl, 20 mm Tris-HCl, pH 8.0, 1 mm EDTA, 0.1% Triton X-100, protease inhibitor mixture (Sigma P4380)) and sonicated. Following centrifugation at 40,000 rpm for 10 min, the cleared lysate was applied to chitin-agarose (New England Biolabs Inc.) pre-equilibrated with lysis buffer, after which the column was washed with 20 column volumes of lysis buffer. Fusion proteins were cleaved on the column overnight by incubation with lysis buffer containing 50 mm dithiothreitol. Eluted fractions containing the bulk of MeCP2 or CDKL5 were pooled. Recombinant purified MeCP2 was dialyzed against the kinase buffer used in the phosphorylation assays whereas CDKL5 was dialyzed against phosphate-buffered saline. Cell Culture and Transfection—The human embryo-derived kidney cell line HEK293 and mouse fibroblasts, NIH3T3, were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum at 37 °C with 5% CO2. Transient transfections were performed with calcium phosphate or Lipofectamine 2000 (Invitrogen) according to the manufacturers instructions and cells harvested 20-36 h post-transfection. In Vitro Phosphorylation Assays—For the detection of CDKL5 autophosphorylation, FLAG-CDKL5 or its mutated derivatives were immunoprecipitated from overexpressing HEK293 cells and incubated with [γ-33P]ATP. Briefly, total cell extracts were prepared from ∼106 transiently transfected cells with high salt lysis buffer (50 mm Tris-HCl, pH 8.0, 500 mm NaCl, 0.1% Nonidet P-40, 1 mm dithiothreitol, phenylmethylsulfonyl fluoride, and protease inhibitor mixture (Sigma P4380)). Equal amounts of protein extract were precleared for 1 h with 20 μl of 100% mouse IgG-agarose beads at 4 °C; 40 μlof 100% EZviewRed Anti-FLAG M2 Affinity gel (Sigma) was added to the cleared extract and the mixture incubated for 2 h at 4 °C. Immunocomplexes were collected by centrifugation, washed five times with the above lysis buffer containing 700 mm NaCl, and 85% of the pellet was used in kinase assays as described previously (8Mari F. Azimonti S. Bertani I. Bolognese F. Colombo E. Caselli R. Scala E. Longo I. Grosso S. Pescucci C. Ariani F. Hayek G. Balestri P. Bergo A. Badaracco G. Zappella M. Broccoli V. Renieri A. Kilstrup-Nielsen C. Landsberger N. Hum. Mol. Genet. 2005; 14: 1935-1946Crossref PubMed Scopus (264) Google Scholar). For the phosphorylation of MeCP2, 3 μg of recombinant hMeCP2E2 were added to the FLAG-CDKL5 pellet resuspended in kinase buffer and subjected to phosphorylation as previously described (8Mari F. Azimonti S. Bertani I. Bolognese F. Colombo E. Caselli R. Scala E. Longo I. Grosso S. Pescucci C. Ariani F. Hayek G. Balestri P. Bergo A. Badaracco G. Zappella M. Broccoli V. Renieri A. Kilstrup-Nielsen C. Landsberger N. Hum. Mol. Genet. 2005; 14: 1935-1946Crossref PubMed Scopus (264) Google Scholar). 85% of the labeled proteins were separated by SDS-PAGE, visualized by autoradiography or quantitated by PhosphorImager analysis (GE Healthcare). To detect the immunopurified CDKL5 derivatives and MeCP2, 15% of the immunocomplexes were assayed by Western blot. A Kodak Image Station 2000R was used to quantitate the signals in the Western blot. CDKL5 autophosphorylation activity was expressed as the ratio between incorporated 33P and the corresponding immunoreactive signal. Fractionated Extracts and Immunoprecipitation Experiments—For fractionation experiments, 5 × 105 HEK293 were collected 36 h after transfection, washed twice with phosphatebuffered saline, and lysed in hypotonic lysis buffer (20 mm Hepes, pH 8.0, 10 mm KCl, 1.5 mm MgCl2, 1 mm EDTA) supplemented with phosphatase and protease inhibitors (1 mm Na3VO4,1mm dithiothreitol, 5 mm NaF, phenylmethylsulfonyl fluoride, and protease inhibitor mixture) by incubation on ice for 45 min. After centrifugation at 500 × g for 5 min the supernatant was kept as the cytosolic fraction. The pelleted nuclei were washed once in the phosphate-buffered saline, resuspended in nuclear lysis buffer (10 mm Hepes, pH 8.0, 1.5 mm MgCl2,1 m KCl, 0.2 mm EDTA, 1% Nonidet P-40, 25% glycerol) supplemented with the above mentioned inhibitors, incubated for 30 min on ice, and subsequently sonicated to destroy genomic DNA. Cellular debris were removed by centrifugation for 30 min at 18,000 × g at 4 °C and the protein extracts analyzed by immunoblotting. For coimmunoprecipitation experiments, 1 × 106 HEK293 cells coexpressing GFP-CDKL5 and FLAG-CDKL5 derivatives were collected, resuspended in lysis buffer (50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 1% Triton, 2 mm EDTA, 10% glycerol) supplemented with protease and phophatase inhibitors (1 mm Na3VO4,1mm dithiothreitol, 5 mm NaF, phenylmethylsulfonyl fluoride, and protease inhibitor mixture), sonicated, and centrifuged for 15 min at 18,000 × g at 4 °C. The extract was cleared and immunoprecipitated with an anti-FLAG resin as described for phophorylation assays and the immunocomplexes washed four times with the lysis buffer and analyzed by Western blot. For the phospho-TEY assay, total extracts from 1 × 106 transiently transfected HEK293 cells were prepared as for coimmunoprecipitation experiments and extracted proteins purified with either anti-FLAG resin as described above or with anti-GFP antibodies. The extracts were incubated for 1 h with the primary antibody, after which protein-G-Sepharose (KPL) was added, the mixture incubated for 2 h and immunocomplexes washed four times with the lysis buffer and analyzed by Western blot. Immunofluorescence—Mouse fibroblasts, NIH3T3, were seeded on gelatin-coated glass coverslips and transiently transfected with Lipofectamine 2000 (Invitrogen). 20 h post-transfection the cells were fixed with 4% paraformaldehyde, stained with Hoechst 33258 (Sigma) and analyzed with an Disease-causing Mutations in in the CDKL5 gene have recently been identified in patients from Rett syndrome, West syndrome, and infantile spasms (2Kalscheuer V.M. Tao J. Donnelly A. Hollway G. Schwinger E. Kubart S. Menzel C. Hoeltzenbein M. Tommerup N. Eyre H. Harbord M. Haan E. Sutherland G.R. Ropers H.H. Ge´cz J. Am. J. Hum. Genet. 2003; 72: 1401-1411Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, J. Van Esch H. Hagedorn-Greiwe M. Hoffmann K. Moser B. Raynaud M. Sperner J. Fryns J.-P. Schwinger E. Ge´cz J. Ropers H.-H. Kalscheuer V.M. Am. J. Hum. Genet. 2004; 75: 1149-1154Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar, 5Weaving L.S. Christodoulou J. Williamson S.L. Friend K.L. McKenzie O.L.D. Archer H. Evans J. Clarke A. Pelka G.J. Tam P.P.L. Watson C. Lahooti H. Ellaway C.J. Bennetts B. Leonard H. Ge´cz J. Am. J. Hum. Genet. 2004; 75: 1079-1093Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar, 6Scala E. Ariani F. Mari F. Caselli R. Pescucci C. Longo I. Meloni I. Giachino D. Bruttini M. Hayek G. Zappella M. Renieri A. J. Med. Genet. 2005; 42: 103-107Crossref PubMed Scopus (209) Google Scholar, 7Evans J.C. Archer H.L. Colley J.P. Ravn K. Nielsen J.B. Kerr A. Williams E. Christodoulou J. Gecz J. Jardine P.E. Wright M.J. Pilz D.T. Lazarou L. Cooper D.N. Sampson J.R. Butler R. Whatley S.D. Clarke A.J. Eur. J. Hum. Genet. 2005; 13: 1113-1120Crossref PubMed Scopus (160) Google Scholar, 8Mari F. Azimonti S. Bertani I. Bolognese F. Colombo E. Caselli R. Scala E. Longo I. Grosso S. Pescucci C. Ariani F. Hayek G. Balestri P. Bergo A. Badaracco G. Zappella M. Broccoli V. Renieri A. Kilstrup-Nielsen C. Landsberger N. Hum. Mol. Genet. 2005; 14: 1935-1946Crossref PubMed Scopus (264) Google Scholar, H.L. Evans J. Edwards S. Colley J. Newbury-Ecob R. O'Callaghan F. Huyton M. O'Regan M. Tolmie J. Sampson J. Clarke A. Osborne J. J. Med. Genet. 2006; 43: 729-734Crossref PubMed Scopus (164) Google Scholar), which the common features of seizures and mental suggesting an important of CDKL5 in the nervous CDKL5 is a large protein of amino acids with an molecular of containing a conserved kinase domain within its terminus and a large COOH-terminal region to which has been (1Montini E. Andolfi G. Caruso A. Buchner G. Walpole S.M. Mariani M. Consalez G. Trump D. Ballabio A. Franco B. Genomics. 1998; 51: 427-433Crossref PubMed Scopus (110) Google Scholar). the of patients is the mutations identified so far are in regions of the protein. missense mutations have been identified in the catalytic domain of the protein; the and mutations have been to with and either kinase activation or substrate (4Tao J. Van Esch H. Hagedorn-Greiwe M. Hoffmann K. Moser B. Raynaud M. Sperner J. Fryns J.-P. Schwinger E. Ge´cz J. Ropers H.-H. Kalscheuer V.M. Am. J. Hum. Genet. 2004; 75: 1149-1154Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar, C. Franco B. Rosner M.R. Hum. Mol. Genet. 2005; 14: 3775-3786Crossref PubMed Scopus (93) Google Scholar). Frameshift mutations the deletion of the protein have been found both in the catalytic domain and in a region spanning almost amino acids of the suggesting an important of this region for CDKL5 functions. To the functional domains of the protein and its are we to CDKL5 derivatives in functional in vitro phosphorylation we also assayed the subcellular as well as the capacity of CDKL5 to interact with itself. We to characterize the two Rett derivatives and mutated within the catalytic domain as well as two truncated proteins Whereas the Δ781 a amino acids deletion identified in a Rett the Δ525 is of of the COOH terminus and the sites within this which binding sites for homology proteins (11Lin C. Franco B. Rosner M.R. Hum. Mol. Genet. 2005; 14: 3775-3786Crossref PubMed Scopus (93) Google Scholar). as a we also analyzed a CDKL5 the entire kinase domain as well as a that has been mutated in the conserved (11Lin C. Franco B. Rosner M.R. Hum. Mol. Genet. 2005; 14: 3775-3786Crossref PubMed Scopus (93) Google Scholar, T. J. PubMed Scopus Google Scholar). Rett Mutations in the CDKL5 catalytic activity of CDKL5 has been to be in its autophosphorylation in vitro (8Mari F. Azimonti S. Bertani I. Bolognese F. Colombo E. Caselli R. Scala E. Longo I. Grosso S. Pescucci C. Ariani F. Hayek G. Balestri P. Bergo A. Badaracco G. Zappella M. Broccoli V. Renieri A. Kilstrup-Nielsen C. Landsberger N. Hum. Mol. Genet. 2005; 14: 1935-1946Crossref PubMed Scopus (264) Google Scholar, C. Franco B. Rosner M.R. Hum. Mol. Genet. 2005; 14: 3775-3786Crossref PubMed Scopus (93) Google Scholar). We to the mutated derivatives for autophosphorylation activity and to this the mutated proteins carrying an were immunopurified from overexpressing cells and the washed pellet incubated with [γ-33P]ATP. 85% of the labeled proteins were separated by and visualized by autoradiography To for the of protein used in these the 15% of the labeled proteins were analyzed by Western using anti-CDKL5 antibodies shown in the in a corresponding to wild-type CDKL5 is the of this in the with that the is specific for CDKL5. Furthermore, the of phosphorylation of the two CDKL5 derivatives catalytic activity to conclude that kinase with CDKL5 its In fact, the corresponding to ΔN and K42R are of signals 3 and The mutation the conserved in and has been to with the catalytic activity of CDKL5 (4Tao J. Van Esch H. Hagedorn-Greiwe M. Hoffmann K. Moser B. Raynaud M. Sperner J. Fryns J.-P. Schwinger E. Ge´cz J. Ropers H.-H. Kalscheuer V.M. Am. J. Hum. Genet. 2004; 75: 1149-1154Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar, C. Franco B. Rosner M.R. Hum. Mol. Genet. 2005; 14: 3775-3786Crossref PubMed Scopus (93) Google Scholar, T. J. PubMed Scopus Google Scholar). In with this hypothesis, we that the is completely of autophosphorylation activity the which is downstream of the phosphorylation within the activation (4Tao J. Van Esch H. Hagedorn-Greiwe M. Hoffmann K. Moser B. Raynaud M. Sperner J. Fryns J.-P. Schwinger E. Ge´cz J. Ropers H.-H. Kalscheuer V.M. Am. J. Hum. Genet. 2004; 75: 1149-1154Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar, T. J. PubMed Scopus Google Scholar), maintains the capacity of autophosphorylation it to have a activity corresponding to of wild-type CDKL5 to as of the last amino acids a autophosphorylation activity to in fact, the Δ781 the wild-type protein and Lin (11Lin C. Franco B. Rosner M.R. Hum. Mol. Genet. 2005; 14: 3775-3786Crossref PubMed Scopus (93) Google have recently that the COOH terminus of CDKL5 has a regulatory a of the kinase corresponding to the catalytic domain had autophosphorylation our data suggest that the COOH-terminal region of the protein to this CDKL5 of the TEY in contains the TEY within its activation this motif has been characterized in the extracellular signal-regulated and its phosphorylation is required for activation of these protein G. F. M. K. 2005; Scholar). of activation is in by however, of the has been shown to be due to its to the TEY motif K. Rosner M.R. J. Full Text Full Text PDF PubMed Scopus Google Scholar). CDKL5 has previously been to be on its TEY in fact, the antibody CDKL5 immunopurified from cells (11Lin C. Franco B. Rosner M.R. Hum. Mol. Genet. 2005; 14: 3775-3786Crossref PubMed Scopus (93) Google Scholar). We