New insights on cellular distribution, microtubule interactions and post-translational modifications of MS-KIF18A.
The present study highlights on the biochemical and immunological analysis of MS-KIF18A in pre-osteogenic MBA-15 cells. The protein distribution in various cellular compartments was demonstrated by imaging and Western blot (WB) analysis. MS-KIF18A interactions with cytoskeletal proteins were confirmed for tubulin and actin. The complex between MS-KIF18A and microtubules (MT) was demonstrated in cellular system for endogenous proteins and also between recombinant proteins in pull down and immunoprecipitation (IP) assays. Multiple assays including metabolic labeling, cell fractionation and IP with anti-MS-KIF18A antibody demonstrated an association with actin that was prominent in the cell cytoplasm. Sub-cellular fractionation identified diverse forms of MS-KIF18A in cytoplasm and membrane/nucleus compartments which are suggested to represent the result of post-transcriptional modifications, such as phosphorylation and glycosylation. These modifications on MS-KIF18A were analyzed by bioinformatics and immunological assays. Furthermore, we studied the role of ubiquitin-proteasome system in the MS-KIF18A degradation. Taken together, the current study sheds light on MS-KIF18A a MT-dependent kinesin and adds insights on the post-translational modifications that potentially control the protein cellular distribution and its co-association with cytoskeletal proteins.
Zusev
Margalit
M
Department of Cell and Developmental Biology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel.
Benayahu
Dafna
D
D000199:Actins / Q000378:metabolism; D002460:Cell Line; D006031:Glycosylation / Q000187:drug effects; D006801:Humans; D016547:Kinesins / Q000378:metabolism*; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism*; D015739:Nocodazole / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D044967:Serum; D014404:Tubulin / Q000378:metabolism
D000199:Actins; D014404:Tubulin; D046988:Proteasome Endopeptidase Complex; C502735:KIF18A protein, human; D016547:Kinesins; D015739:Nocodazole