Affiliation:
1. Howard Hughes Medical Institute, Department of Cardiology, Department of Neurobiology, Harvard Medical School, 1309 Enders Building, 320 Longwood Avenue, Children's Hospital, Boston, MA 02115, USA.
Abstract
We cloned and characterized a protein kinase and ion channel, TRP-PLIK. As part of the long transient receptor potential channel subfamily implicated in control of cell division, it is a protein that is both an ion channel and a protein kinase. TRP-PLIK phosphorylated itself, displayed a wide tissue distribution, and, when expressed in CHO-K1 cells, constituted a nonselective, calcium-permeant, 105-picosiemen, steeply outwardly rectifying conductance. The zinc finger containing α-kinase domain was functional. Inactivation of the kinase activity by site-directed mutagenesis and the channel's dependence on intracellular adenosine triphosphate (ATP) demonstrated that the channel's kinase activity is essential for channel function.
Publisher
American Association for the Advancement of Science (AAAS)
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4. Cloning of PLIK: Sequence shown in supplementary material (22). RACE experiments were performed to determine the open reading frame of PLIK and its 5′ untranslated region (UTR) with rat brain Marathon cDNA (Clontech). Description of the primers and conditions for cloning is available upon request.
5. Cloning of mouse TRP-PLIK TRP-PLIK-HA TRP-PLIK mutants and GST-kinase and mutants: TRP-PLIK was cloned directly by PCR with primers based on the deposited sequence of ChaK (nucleotides 95 to 6333) by M. Matsushita (accession number: ). Description of the primers and conditions for cloning is available upon request. TRP-PLIK lacks a CAG codon for alanine at residue 1494 of ChaK which is most likely due to a polymorphism. To make TRP-PLIK-HA we first introduced a Cla I site into TRP-PLIK by exchanging the last two codons (ATA GAG to ATC GAT) using the QuikChange site-directed mutagenesis kit (Stratagene). A synthetic oligonucleotide encoding the HA tag (YPYDVPDYA) was then subcloned into the Cla I site. QuikChange was also used to make the ATP-mut substitution (GGA to GAT) and the Zn-mut double substitution (TGT to GCT and TGC to GCC). The “kinase domain” of TRP-PLIK (residues 1532 to 1862) was directionally subcloned in frame with GST into the Bam HI and Eco RI sites of the pGEX-6P-3 vector. GST and GST-fusion proteins were expressed in BL21-CodonPlus(DE3)-RP strain of E. coli (Stratagene).
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