Neurokinin Receptors

The transcript coding isoform 2 lacks exons 4 and 13, and its translation starts from an internal ATG codon in a different reading frame than isoform 1

The transcript coding isoform 2 lacks exons 4 and 13, and its translation starts from an internal ATG codon in a different reading frame than isoform 1. similarly to it, was speculated to inhibit Cdk5 (4) and Banoxantrone D12 thereby affect exocytosis of synaptic vesicles (5) and insulin granules (6C9). This hypothesis was confuted, however, as CDKAL1 was repeatedly shown neither to interact with Banoxantrone D12 Cdk5 nor inhibit its activity (10, 11). Meanwhile, independent work led to the discovery that is a methylthiotransferase responsible for the modification of tRNAs (12). This modification was shown to improve codon recognition and accuracy of reading frame maintenance (13), thus ensuring fidelity during protein synthesis. Remarkably, CDKAL1, which displays sequence similarity with YqeV, was able to rescue this enzymatic activity in a strain. Wei (11) elegantly demonstrated that CDKAL1 is indeed a methylthiotransferase that specifically modifies tRNALys in mammals. The lack of CDKAL1 caused misreading of the Lys codon, and amino acids were erroneously incorporated during translation, resulting in aberrant proteolytic processing of proinsulin and a probable increase in its degradation. Here, we show that down-regulation of CDKAL1 in insulinoma cells perturbs levels of not only insulin but also chromogranin A and islet cell autoantigen 512 (ICA512/IA-2), two other components of insulin secretory granules. We additionally show that CDKAL1 is a new tail-anchored (TA) protein that exploits the TRC40/Get3 pathway for its insertion in the ER membrane. Finally, we report that CDKAL1 mRNA is up-regulated upon induction of the unfolded protein response, further suggesting a role for CDKAL1 in the regulation of the secretory pathway. EXPERIMENTAL PROCEDURES Cell Culture and Transfection Rat insulinoma INS-1 cells were grown and electroporated as described (14). Human pancreatic islets isolated from an organ donor with written consent for research use were kindly provided by Barbara Ludwig and Stefan Bornstein (Medical Clinic III, University Clinic, Dresden University of Technology). Cloning of Rat/Human CDKAL1 cDNA RNA was isolated from INS-1 cells and human pancreatic islets with an RNeasy mini kit (Qiagen); reverse transcription of 2 g of RNA was performed with Superscript II reverse transcriptase (Invitrogen) using oligo(dT). cDNA was then amplified with primers annealing to 5/3-UTRs, based on human SMOC2 Ref Seq “type”:”entrez-nucleotide”,”attrs”:”text”:”NM_017774.3″,”term_id”:”291084722″,”term_text”:”NM_017774.3″NM_017774.3 and rat Ref Seq “type”:”entrez-nucleotide”,”attrs”:”text”:”XM_341524.3″,”term_id”:”109504794″,”term_text”:”XM_341524.3″XM_341524.3, which has in the meantime been discontinued (rat 5-UTR, 5-tttcctgacagtggcttgtg-3; rat 3-UTR, 5-tgagaactggtgctgttgct-3; human 5-UTR, 5-tagactaattgcagataattaagag-3; human 3-UTR, 5-ctttatagatgtttccattagttg-3). PCR products were cloned into the pCRII vector by TA cloning (Invitrogen) and verified by DNA sequencing. The sequence of rat CDKAL1 cDNA was scanned against the database, and no other genes with significant relatedness were identified. Plasmids All constructs used here were made by standard recombinant DNA techniques and verified by sequencing. Full-length human CDKAL1 cDNA (IMAGE clone 40117357) was purchased from Open Biosystems. Its open reading frame was amplified by PCR, flanked with HindIII and XbaI restriction sites, and subcloned in-frame with three HA epitopes at its C terminus, as described (15). pGEM4-hCDKAL1-Nglyc was generated by amplifying human CDKAL1 with the following primers: fw-KpnI 5-ctcggtaccatgccttctgcatcctg-3 and rev-BspEI 5-tttgtcaaggtctataattccggaat-3. The KpnI-BspEI-digested PCR product replaced the cytochrome translation of rat CDKAL1 in pCRII was performed with the TnT-T7 Quick-coupled Transcription/Translation System (Promega) according to the manufacturer’s protocol. Reactions performed in the presence of [35S]methionine were analyzed by SDS-PAGE and phosphorimaging (BAS 1800II phosphorimager (Fuji)); reactions performed with nonradioactive methionine were resolved by SDS-PAGE and analyzed by immunoblot. Transcription and translation of hCDKAL1-Nglyc, to recover the post-nuclear supernatant. The latter was separated in a high Banoxantrone D12 speed supernatant and a high speed pellet (HSP) by centrifugation at 4 C for 30 min at 150,000 in a Beckman TLA100.1 rotor. The HSP was resuspended in HB buffer (half of the starting homogenate volume), treated with an equal volume of 0.2 m Na2CO3 at pH 11.5 for 30 min on ice, and then brought Banoxantrone D12 to 1.5 m sucrose, 0.1 m Na2CO3 in a final volume of 0.5 ml. The sample was layered under a discontinuous sucrose gradient composed of 2.