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Regulation of the CDK-related protein kinase PCTAIRE-1 and its possible role in neurite outgrowth in Neuro-2A cells

Ralph Graeser^1,*,, Julian Gannon¹, Randy Y. C. Poon², Thierry Dubois^3,, Alastair Aitken³ and Tim Hunt¹

¹ ICRF Clare Hall Laboratories, South Mimms, Herts EN6 3LD, UK
² Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong
³ Division of Biomedical and Clinical Laboratory Sciences, Section of Biomedical Sciences, The University of Edinburgh, Hugh Robson Building, George Square, Edinburgh EH8 9XD, UK
^* Present address: Target Identification and Validation, ProQinase GmbH, Breisacher Str. 117, D-79106 Freiburg, Germany
Present address: Institut Curie — Section Recherche, CNRS UMR 144, 26 rue d'Ulm, 75248 Paris Cedex 05, France

View larger version (43K): [in a new window]   Fig. 1. Activation and phosphorylation of recombinant bacterially expressed PCTAIRE-1 in brain and testis extracts. (A) Bacterial lysates containing either wild-type or kinase-dead myc-tagged PCTAIRE-1 proteins were incubated in brain or testis extracts or buffer, and the bound material was assayed for protein kinase activity towards myelin basic protein (MBP). (top panel) Coomassie-blue-stained gel; (middle panel) autoradiograph at the position of PCTAIRE-1; (bottom panel) autoradiograph at the position of MBP. (B) Purified wild-type and truncated recombinant myc-tagged PCTAIRE-1 proteins were incubated in brain and testis extracts or buffer, immunoprecipitated, and incubated in the presence of [-32P]ATP. (top panel) Coomassie-blue-stained gel; (bottom panel) autoradiograph at the position of PCTAIRE-1.

View larger version (23K): [in a new window]   Fig. 2. PKA phosphorylates PCTAIRE-1. (A) Elution profile of labeled tryptic peptides of PCTAIRE-1, prepared as described in Materials and Methods. (B) Wild-type, or mutated recombinant PCTAIRE-1 proteins were tested as PKA substrates using 1 U of purified PKA. (top panel) Coomassie-blue-stained gel; (bottom panel) autoradiograph at the position of PCTAIRE-1.

View larger version (23K): [in a new window]   Fig. 3. Phosphorylation of S153 by PKA inhibits PCTAIRE-1 kinase activity. (A) Constructs encoding wild-type or mutated YFP-tagged PCTAIRE-1 were transfected into Neuro-2A cells. 48 hours after transfection, the cells were lysed, the YFP-tagged PCTAIRE-1 proteins immunoprecipitated using a rabbit serum against GFP, and the bound material was assayed for kinase activity towards MBP. (top panel) Coomassie-blue-stained gel; (middle panel) autoradiograph at the position of MBP; (bottom panel) radioactivity (cpm) in the MBP bands. (B) Constructs encoding wild-type and YFP-tagged PCTAIRE-1 were transfected into Neuro-2A cells. 48 hours after transfection, the cells were treated with 10 µM forskolin to stimulate PKA for 2 hours, or left untreated. Cells were lysed, the YFP-tagged PCTAIRE-1 proteins were immunoprecipitated using a rabbit serum against GFP and assayed for kinase activity towards MBP. (top panel) Coomassie-blue-stained gel; (middle panel) autoradiograph at the position of MBP; (bottom panel) radioactivity (cpm) in the MBP bands. (C) Constructs encoding wild-type and mutated YFP-tagged PCTAIRE-1 were transfected into Neuro-2A cells. 48 hours after transfection, the cells were lysed, the YFP-tagged PCTAIRE-1 proteins immunoprecipitated using a rabbit serum against GFP, and the bound material was incubated in the presence or absence of PKA, washed, and then assayed for kinase activity towards MBP. (top panel) Coomassie blue stained gel; (middle panel) autoradiograph at the position of MBP; (bottom panel) radioactivity (cpm) in the MBP bands.

View larger version (48K): [in a new window]   Fig. 4. PCTAIRE-1 kinase activity — substrates and structural requirements. (A) Neuro-2A cells were transfected with S153A mutated YFP-tagged PCTAIRE-1. 48 hours after transfection, the cells were lysed, the YFP-tagged PCTAIRE-1 proteins immunoprecipitated using a rabbit serum against GFP, and the bound material was assayed for kinase activity towards various substrates. (top panel) Coomassie-blue-stained gel; (middle panel) autoradiograph at the position of MBP; (bottom panel) radioactivity (cpm) in the substrate bands. (B) Neuro-2A cells were transfected with constructs encoding wild-type, mutated and truncated YFP-tagged PCTAIRE-1. Two days after the transfection, the cells were lysed and the YFP-tagged PCTAIRE-1 proteins were immunoprecipitated using a rabbit serum against GFP, washed, and assayed for kinase activity towards MBP. (top panel) Coomassie-blue-stained gel; (second panel) autoradiograph at the position of PCTAIRE-1; (third panel) autoradiograph at the position of MBP; (bottom panel) radioactivity (cpm) in the MBP bands. (C) Neuro-2A cells were transfected with constructs encoding N-terminally truncated, kinase-dead and S153A mutated YFP-tagged PCTAIRE-1. Two days after the transfection, the cells were lysed, and the YFP-tagged PCTAIRE-1 proteins were immunoprecipitated using a rabbit serum against GFP, washed, and assayed for kinase activity towards MBP. (top panel) Coomassie-blue-stained gel; (middle panel) autoradiograph at the position of MBP; (bottom panel) radioactivity (cpm) in the MBP bands.

View larger version (41K): [in a new window]   Fig. 5. Does PCTAIRE-1 require a brain-specific partner protein? (A) Gel filtration analysis of an extract from mouse brain. The lysate prepared from one mouse brain was applied on an AcA34 column, PCTAIRE-1 was immunoprecipitated from the individual fractions using mAb G6, and assayed for kinase activity. The reaction mix was then applied on an SDS-PAGE, blotted, and checked with a polyclonal PCTAIRE-1 antibody for the presence of PCTAIRE-1. Subsequently, the blot was exposed for autoradiography. (B) Constructs encoding wild-type, kinase-dead, or S153A mutated YFP-tagged PCTAIRE-1 were transfected into Neuro-2A, HEK 293, CHO, COS-7 and HeLa cells. Two days after the transfection, the cells were lysed, and the YFP-tagged PCTAIRE-1 proteins were immunoprecipitated using a rabbit serum against GFP, and assayed for kinase activity on MBP. (top panel) Coomassie-blue-stained gel; (middle panel) autoradiograph at the position of MBP; (bottom panel) radioactivity (cpm) in the MBP bands. The S153A mutant PCTAIRE-1 protein kinase is active in a wide range of cell lines. (C) Constructs encoding wild-type, kinase-dead, S153A, S119A/S153A mutated YFP-tagged PCTAIRE-1 and YFP-tagged CDK5, were transfected into Neuro-2A and HEK 293 cells. Two days after the transfection, the cells were lysed, and the YFP-tagged proteins were immunoprecipitated using a rabbit serum against GFP, and assayed for kinase activity towards MBP. p21nck was added to reactions with CDK5 as indicated. (top panel) Coomassie-blue-stained gel; (middle panel) autoradiograph at the position of MBP; (bottom panel) radioactivity (cpm) in the MBP bands. Note that PCTAIRE-1 and CDK5/p21nck display comparable kinase activity.

View larger version (40K): [in a new window]   Fig. 6. Phosphorylation of S119 by PKA generates a 14-3-3 binding motif. (A) Wild-type and S119A mutated recombinant PCTAIRE-1 proteins were bound to 9E10 beads, incubated either in the presence or absence of 1U PKA, followed by incubation with GST-tagged 14-3-3 or isoforms. The beads were washed, and the bound material was analysed by western blotting using antisera against PCTAIRE-1 (top panel) and GST (bottom panel). In lanes 5 and 6, 10% of the input of the respective 14-3-3 proteins were applied. (B) Constructs encoding wild-type or mutated YFP-tagged PCTAIRE-1 were transfected into Neuro-2A cells. Two days after the transfection, the cells were lysed, the YFP-tagged PCTAIRE-1 proteins were immunoprecipitated using a rabbit serum against GFP, and the bound material was checked for the presence of 14-3-3 proteins and PCTAIRE-1 by western blotting using antisera against 14-3-3-proteins (pan) and PCTAIRE-1, respectively.

View larger version (61K): [in a new window]   Fig. 7. Morphological effects of PCTAIRE-1-YFP constructs transfected into Neuro-2A cells suggest a role of PCTAIRE-1 in the prevention of neurite outgrowth. (A) Indicated constructs were transfected into Neuro-2A cells. Two days after transfection, the individual wells were scored for green cells that had neurites longer than twice the length of the cell body. The cells were then treated with 10 µM forskolin, left for 24 hours, and scored again. 5-10 fields were counted totalling 500-1000 transfected cells. The experiment was repeated twice with similar results. (B) Images of cells transfected with YFP, or wild-type, kinase-dead, or the double mutant S119/S153-YFP-PCTAIRE-1 constructs, showing the effects on neurite outgrowth.

View larger version (61K): [in a new window]   Fig. 8. Nuclear targeting of PCTAIRE-1 abolishes its effects on neurite outgrowth. (A) Indicated constructs were transfected into Neuro-2A cells together with a CD2 marker construct. Two days after transfection, the individual wells were treated with 10 µM forskolin and left for another 24 hours. The wells were incubated for 5 minutes using a Cy3 coupled CD2 antibody, and scored for green/red cells that had neurites longer than twice the length of the cell body. (B) Images of the transfected cells showing the effects on neurite outgrowth.

View larger version (18K): [in a new window]   Fig. 9. Schematic representation of PCTAIRE-1 compared with PCTAIRE-2 and -3. (A) On the left-hand side of the sketch the three known PCTAIREs are aligned with their catalytic kinase domain represented as a grey box. On the right hand side, PCTAIRE-1 is depicted again with regions of high homology between all three genes in a black box, and those between PCTAIRE-1 and -2 in a grey box. Additionally, the ATP-binding site and the T-loop residue, as well as the residues phosphorylated by PKA, are shown along with their function for PCTAIRE-1. (B) Alignment of the PCTAIRE-1 region surrounding the residues involved in 14-3-3 binding, S119 and S153, with a dimeric 14-3-3 peptide inhibitor [difopein (Masters and Fu, 2001)]. The position of S119 and S153 are marked with asterisks. Identical residues are connected with a bar, and related residues with a colon.