Molecular and functional characterization of a novel splice variant of ANKHD1 that lacks the KH domain and its role in cell survival and apoptosis.
Multiple ankyrin repeat motif-containing proteins play an important role in protein-protein interactions. ANKHD1 proteins are known to possess multiple ankyrin repeat domains and a single KH domain with no known function. Using yeast two-hybrid system analysis, we identified a novel splice variant of ANKHD1. This splice variant of ANKHD1, which we designated as HIV-1 Vpr-binding ankyrin repeat protein (VBARP), does not contain the signature KH domain, and codes for only a single ankyrin repeat motif. We characterized VBARP by molecular and functional analysis, revealing that VBARP is ubiquitously expressed in different tissues as well as cell lines of different lineage. In addition, blast searches indicated that orthologs and homologs to VBARP exist in different phyla, suggesting that VBARP might be evolutionarily conserved, and thus may be involved in basic cellular function(s). Furthermore, biochemical analysis revealed the presence of two VBARP isoforms coding for 69 and 49 kDa polypeptides, respectively, that are primarily localized in the cytoplasm. Functional analysis using short interfering RNA approaches indicate that this gene product is essential for cell survival through its regulation of caspases. Taken together, these results indicate that VBARP is a novel splice variant of ANKHD1 and may play a role in cellular apoptosis (antiapoptotic) and cell survival pathway(s).
Miles
Melissa C
MC
Department of Infectious Diseases & Microbiology, Graduate School of Public Health, University of Pittsburgh, PA 15261 , USA.
Janket
Michelle L
ML
Wheeler
Elizabeth D A
ED
Chattopadhyay
Ansuman
A
Majumder
Biswanath
B
Dericco
Jeremy
J
Schafer
Elizabeth A
EA
Ayyavoo
Velpandi
V
D000595:Amino Acid Sequence; D017487:Ankyrins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D017209:Apoptosis / Q000502:physiology*; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival / Q000502:physiology*; D017931:DNA Primers; D019084:Fluorescent Antibody Technique, Indirect; D016336:Gene Products, vpr / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding; D012326:RNA Splicing; D034741:RNA, Small Interfering / Q000378:metabolism; D016601:RNA-Binding Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013347:Subcellular Fractions / Q000378:metabolism; D020798:Two-Hybrid System Techniques
C504171:ANKHD1 protein, human; D017487:Ankyrins; D002352:Carrier Proteins; D017931:DNA Primers; D016336:Gene Products, vpr; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D020169:Caspases