Regulation of protein kinase CK1alphaLS by dephosphorylation in response to hydrogen peroxide

Abstract

Low levels of hydrogen peroxide (H(2)O(2)) are mitogenic to mammalian cells and stimulate the hyperphosphorylation of heterogeneous nuclear ribonucleoprotein C (hnRNP-C) by protein kinase CK1alpha. However, the mechanisms by which CK1alpha is regulated have been unclear. Here it is demonstrated that low levels of H(2)O(2) stimulate the rapid dephosphorylation of CK1alphaLS, a nuclear splice form of CK1alpha. Furthermore, it is demonstrated that either treatment of endothelial cells with H(2)O(2), or dephosphorylation of CK1alphaLS in vitro enhances the association of CK1alphaLS with hnRNP-C. In addition, dephosphorylation of CK1alphaLS in vitro enhances the kinase's ability to phosphorylate hnRNP-C. While CK1alpha appears to be present in all metazoans, analysis of CK1alpha genomic sequences from several species reveals that the alternatively spliced nuclear localizing L-insert is unique to vertebrates, as is the case for hnRNP-C. These observations indicate that CK1alphaLS and hnRNP-C represent conserved components of a vertebrate-specific H(2)O(2)-responsive nuclear signaling pathway.

Fig. 1

CK1αLS is Dephosphorylated in Response to H₂O₂. (a) 1-dimensional immunoblots of the crude HUVEC nuclear / plasma membrane fraction showing proteins containing the L-insert and the S-insert of CK1α. CK1αLS was identified as a ~35 kDa protein reactive to both L-insert and S-insert specific antibodies. CK1αS was identified as a ~32 kDa protein reactive only to the S-insert specific antibodies. (b) 2-dimensional immunoblots of HUVEC nuclear extracts probing for CK1αLS using L-insert specific antibodies. The scale at the top refers to the apparent isoelectric point. Under resting conditions, CK1αLS is present predominantly with a isoelectric point between 8.7 and 9.0. Treatment with 5 µM H₂O₂ for 15 min shifts the isoelectric point of CK1αLS to 9.0–9.4. The same change is seen upon incubation of the nuclear extracts with alkaline phosphatase (AP). (c) HUVECs metabolically labeled with ³²P were treated with or without 5 µM H₂O₂ for 15 min. CK1αLS was immunoprecipitated from nuclear extracts and assessed for ³²P incorporation by phosphorescence imaging and for total CK1αLS protein by immunoblotting using L-insert specific antibodies. All blots are representative of at least 3 independent experiments.

Fig. 2

CK1αLS Dephosphorylation Occurs Rapidly in Response to Low Levels of H₂O₂. Panels on the left are portions of 2-dimensional immunoblots of HUVEC nuclear extracts probing for CK1αLS using L-insert specific antibodies. The scales at the top refer to the isoelectric point. Dephosphorylation of CK1αLS was assessed by the shift to an isoelectric point > 8.9. The panels on the right depict quantitation of the changes from 3 independent experiments. Error bars indicate the standard deviation. (a) A time course analysis using 5 µM H₂O₂ indicates that the H₂O₂–induced dephosphorylation of CK1αLS is first apparent at 5 min after treatment and nearly complete by 10 min after treatment. ** P < 0.001 versus time 0 min. (b) Varying the concentration of applied H₂O₂ and assessing 15 min after treatment reveals substantial dephosphorylation of CK1αLS with just 1 µM H₂O₂, with the effect being maximal with 5 µM H₂O₂. ** P < 0.001 versus no H₂O₂ added.

Fig. 3

Dephosphorylation of CK1αLS Promotes Its Association with hnRNP-C. (a) HUVECs were treated with or without 1 µM H₂O₂ for 10 min. CK1αLS was immunoprecipitated from nuclear extracts using S-insert specific antibodies. The immunoprecipitates were subjected to immunoblotting for hnRNP-C and also for CK1αLS using L-insert specific antibodies. (b) CK1αLS was immunoprecipitated from the nuclear extracts of untreated HUVECs using S-insert specific antibodies and then incubated in vitro with or without alkaline phosphatase (Alk. Phos.). The immunopurified kinase was then added back to the nuclear extracts to associate with hnRNP-C. The complexes were immunoprecipitated and subjected to immunoblotting for hnRNP-C and also for CK1αLS using L-insert specific antibodies. (c) Quantitation of the experiments in (a) and (b) using three independent experiments. Error bars indicate the standard deviation; ** P<0.01 versus controls; ie no H₂O₂ for the comparison on the left and no Alk. Phos. for the comparison on the right.

Fig. 4

Dephosphorylation of CK1αLS Enhances the Phosphorylation of hnRNP-C by the Kinase. CK1αLS immunoprecipitated from HUVEC nuclei was treated with or without alkaline phosphatase (Alk. Phos.) to remove all phosphate groups. The kinase was then incubated with intact hnRNP-C tetramers immunoprecipitated from HUVEC nuclei, and the phosphorylation status of hnRNP-C was assessed by (a) ³²P incorporation into hnRNP-C and also by (b) 2-dimensional immunoblotting for hnRNP-C. Dephosphorylation of CK1αLS enhanced the phosphorylation of hnRNP-C and specifically enhanced the formation of hyperphosphorylated forms of hnRNP-C, which have an isoelectric point of 4.9. All blots are representative of at least 3 independent experiments.

Fig. 5

CK1αLS is a Vertebrate-Specific Nuclear Kinase. (a) Depicted is an alignment of L-insert sequences identified in multiple vertebrate species as well as the L-insert like sequence from Saccharomyces cerevisiae Yck3. Green shading indicates completely invariant residues. Yellow shading indicates residues invariant throughout vertebrates but differing in yeast, and the red shading indicates the residues that differ from the human L-insert sequence. The accession numbers for the sequences are: Human, AADC01055869; Chimpanzee, AACZ020650861; Rhesus Monkey, AANU01185333; Cow, AAFC03126961; Elephant, AAGU01149089; Hedgehog, AAIY01297366; Dog, AAEX02012126; Opossum, AAFR03012999; Shrew, AALT01221290; Chicken, AADN02074969; Frog, EB476829; Mouse, AAHY01139286; Rat, AAHX01095469; Zebrafish, NC_007125; Goldfish, AB120746; Tiger Puffer Fish, CAAB01002152; Stickelback, DV014751; Medaka, BAAF02059097; and Yeast (Yck3), X87108. (b) An analysis of the intron/exon boundaries in the CK1α genes from several species in the region of the L-insert is shown. For all genes, the numbering is based on the human CK1α protein sequence. For numbering purposes, immediately adjacent to non-phase 0 introns, the amino acid was assigned to the exon containing 2 of the 3 nucleotides of the codon. Exons are depicted by thick black bars and are drawn to scale. The exon encoding the L-insert is shown in red. The introns are depicted by thin black lines and are not drawn to scale. Accession numbers are listed in the Experimental Procedures section.