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CrkRS

a novel conserved Cdc2-related protein kinase that colocalises with SC35 speckles

Wellcome/CRC Institute, Tennis Court Road, Cambridge, CB2 1QR, UK
^* Author for correspondence (e-mail: j.pines{at}welc.cam.ac.uk )

View larger version (74K): [in a new window]   Fig. 1. CrkRS is a novel CDK-related protein kinase. (A) Nucleotide and predicted amino acid sequences of the CrkRS cDNA. CrkRS cDNA has an open reading frame of 4473 nucleotides encoding 1490 amino acids. The RS domain is in bold and the kinase domain is underlined. Residues corresponding to the conserved threonine and tyrosine phosphorylation sites of CDK1 are in bold and underlined. A run of nine proline residues is in italics. These sequence data are available from GenBank/EMBL/DDBJ, accession number AF227198. (B) Alignment of the amino^Macid residues encompassing the kinase domains of CHED, CrkRS, and the gene^Mproducts of CG7597 (Drosophila) and B0285 (C. elegans). The^MCHED, CG7597, B0285.1 and B0285.2 sequences were obtained from GenBank^M(accession numbers A38197, AAF51738, CAA84302 and Z34533, respectively). Note,^Mthe C. elegans protein kinase is currently split into two genes in^Mthe database. The RS and proline-rich domain on clone B0285.2 was removed from^Mthe protein kinase domain on clone B0285.1 by the annotators because it did^Mnot resemble any CDKs in the database at that time (R. Durbin, personal^Mcommunication). (C) Schematic diagrams of CrkRS and the gene products of^MCG7597 (Drosophila) and B0285 (C. elegans). Bipartite^Mnuclear localisation sequences (NLS) are indicated by arrows, RS domains by^Mblack rectangles, kinase domains by grey rectangles, PEST regions by triangles^Mand SH3-binding domains by squares.

View larger version (49K): [in a new window]   Fig. 2. CrkRS is ubiquitously expressed and encoded by a single gene. (A) Multiple human tissue northern blots were probed with either the 3'UTR or the kinase domain of CrkRS. Two transcripts, of 6.8 kb and 8.5 kb in length, were recognised in all tissues tested with both probes. RNA size markers are indicated on the left. (B). Southern blot of human genomic DNA probed with the CrkRS 3'UTR probe. Each lane contained 10 µg of DNA digested, individually, with the restriction enzyme indicated. DNA size markers (kb) are indicated on the left.

View larger version (7K): [in a new window]   Fig. 3. CrkRS has an apparent Mr of 180 kDa on SDS-PAGE. (A) Affinity-purified anti-CrkRS antibodies are specific for CrkRS. Immunoblot of the GST-CrkRS C-terminal fragment (GST-CCT) fusion protein purified from E. coli and probed with affinity purified anti-CrkRS antibodies. The antibodies did not recognise the lane containing only GST (i), and pre-clearing the anti-CrkRS antiserum abolished the CrkRS signal (ii). (B) (His)6-tagged human CRKRS expressed in Sf9 cells runs at 180 kDa. Anti-CrkRS immunoblot of lysates from Sf9 cells either uninfected, or infected with wild-type, or CrkRS-expressing baculovirus. (C) In vitro transcription/translation product of the CrkRS cDNA has an apparent Mr of 180 kDa. Full-length CrkRS was transcribed and translated in vitro from an expression vector containing the full length CrkRS cDNA (lane 2). An empty expression vector was used as a control (lane 1).

View larger version (15K): [in a new window]   Fig. 4. CrkRS is a nuclear protein that is phosphorylated in a cell-cycle-dependent manner. (A) CrkRS is a nuclear protein. Immunoblots of cytosolic and nuclear fractions of HeLa cell lysate probed with anti-CrkRS antibodies (lanes 1,2) or precleared anti-CrkRS antibodies (lanes 3,4). (B) Anti-CrkRS immunoprecipitates have protein kinase activity. CrkRS was immunoprecipitated with anti-CRKRS antibodies and assayed for kinase activity in vitro. CrkRS (+) and an unidentified 85 kDa band (++) were phosphorylated in all anti-CrkRS immunoprecipitates (lanes 3). SR-type splicing factor ASF, casein, histone HI, myelin basic protein (MBP) and the GST-tagged CTD of yeast RNA polymerase II were added as exogenous substrates. Arrowheads indicate the position of the exogenous substrate. Kinase buffer alone (lanes 1)and anti-rabbit IgG immunoprecipitates (lanes 2) were assayed as controls. (C) CrkRS levels do not vary during the cell cycle. HeLa cells were synchronised by a thymidine/aphidicolin regime, released and collected at various times to enrich for different cell cycle phases; G1 phase (G1), S phase (S), G2 phase (G2), mitosis (M), and mitotic cells arrested with nocdazole (M*). Fractions were immunoblotted with anti-CrkRS antibodies. Note that CrkRS levels do not vary but CrkRS migrates more slowly on SDS-PAGE at mitosis. (D) CrkRS is a phosphoprotein. Anti-CrkRS immunoprecipitates were treated, or not, with protein phosphatase, in the absence or presence of phosphatase inhibitors and immunoblotted with anti-CrkRS antibodies. Note that both interphase (I) and mitotic (M) forms of CrkRS shift downwards after phosphatase treatment.

View larger version (23K): [in a new window]   Fig. 5. CrkRS localises to SC35 nuclear speckles. (A) HeLa cells were fixed and immunostained with affinity-purified anti-CrkRS antibodies (i), precleared affinity-purified anti-CrkRS antibodies (ii) or control rabbit IgG (iii) and visualised by confocal laser scanning microscopy. (B) CrkRS mainly colocalises with SC35. To verify that CrkRS localises to nuclear speckles, cells were costained with anti-CrkRS antibodies and antibodies against SC35, a known nuclear speckle protein. The merged image shows the extent of overlap between CrkRS and SC35.

View larger version (24K): [in a new window]   Fig. 6. CrkRS strongly associates with nuclear speckles. (A) CrkRS remains associated with nuclear speckles after DNase and RNase treatment. Hela cells were fixed and left untreated (top row) or treated with DNase 1 (middle row) or with RNase A (bottom row). Cells were costained with anti-CrkRS (green), anti-snRNPs (Y12, red) and a DNA marker (TOTO-3, blue). The localisations of CrkRS and snRNPs did not alter significantly in DNase I-treated cells. In RNase A-treated cells, the localisation of CrkRS did not alter significantly but the anti-snRNP staining was less punctate and more uniformly distributed throughout the nuclei. (B) -amanitin alters the pattern of both CrkRS and SC35. In HeLa cells treated with -amanitin both anti-CrkRS (left) and SC35 immunofluorescence patterns (middle) alter to one of fewer, larger and rounder speckles and these colocalise (merged image). (C) Localisation of CrkRS during metaphase and telophase. At metaphase (top), anti-CrkRS (left) and anti-SC35 (middle) immunofluorescence patterns become diffusely distributed throughout the cells, and do not stain the chromosomes (right). At telophase (bottom), anti-CrkRS antibodies and anti-SC35 both stain the reforming nuclear speckles.

View larger version (30K): [in a new window]   Fig. 7. CrkRS is an MPM-2 antigen. (A) Immunoblot. Anti-CrkRS immunoprecipitates from interphase and mitotic HeLa cell fractions were incubated in buffer alone, with protein phosphatase, or with protein phosphatase plus phosphatase inhibitors, and immunoblotted with the MPM-2 mAb (top) or with anti-CrkRS antibodies (bottom). CrkRS was recognised by MPM-2 mAbs in a phosphorylation-dependent manner. (B) CrkRS and MPM-2 colocalise during interphase. HeLa cells were fixed and stained with MPM-2 mAb (left) and anti-CrkRS antibodies (middle). The patterns overlap in the nuclear speckles (Merge).

View larger version (25K): [in a new window]   Fig. 8. The RS domain of CrkRS is required for its localisation to nuclear speckles. (A) Schematic diagram of the CrkRS deletion constructs (i-x). The YFP tag (green), kinase domain (red) proline-rich regions (blue), RS domain (grey) and the bipartite nuclear localisation sequences (stars) are shown. Constructs (i-x) were microinjected into HeLa cells and the subcellular localisation of each fusion protein assayed by confocal laser scanning microscopy. (B) Representative fluorescence images. Each image is representative of 60 microinjected cells 4-6 hours after microinjection. Constructs were assayed twice in two separate experiments.