Ruk is ubiquitinated but not degraded by the proteasome.
The regulator of ubiquitous kinase (Ruk) protein, also known as CIN85 or SETA, is an adaptor-type protein belonging to the CD2AP/CMS family. It was found in complexes with many signaling proteins, including phosphoinositol (PtdIns) 3-kinase (EC 2.7.1.137), Cbl, GRB2, p130Cas and Crk. Functional analysis of these interactions, implicated Ruk in the regulation of apoptosis, receptor endocytosis and cytoskeletal rearrangements. We have recently demonstrated that overexpression of Ruk induces apoptotic death in neurons, which could be reversed by activated forms of PtdIns 3-kinase and PKB/Akt. Furthermore, Ruk was shown to be a negative regulator of PtdIns 3-kinase activity through binding to its P85 regulatory subunit [Gout, I., Middleton, G., Adu, J., Ninkina, N. N., Drobot, L. B., Filonenko, V., Matsuka, G., Davies, A.M., Waterfield, M. & Buchman, V. L. (2000) Embo J.19, 4015-4025]. Here, we report for the first time, that all three isoforms of Ruk (L, M and S) are ubiquitinated. Specific interaction between the E3 ubiquitin ligase Cbl and all three Ruk isoforms was demonstrated by coexpression studies in Hek293 cells. The interaction of Ruk M and S isoforms with Cbl was found to be mediated via heterodimerization with Ruk L. The use of proteosomal and lysosomal inhibitors clearly indicated that ubiquitination of Ruk L does not lead to its degradation. Based on this study, we propose a possible mechanism for the regulation of Ruk function by ubiquitination.
Verdier
Frédérique
F
Ludwig Institute for Cancer Research, University College of London Medical School, London, UK. verdier@cochin.inserm.fr
Valovka
Taras
T
Zhyvoloup
Alexander
A
Drobot
Ludmila B
LB
Buchman
Vladimir
V
Waterfield
Mike
M
Gout
Ivan
I
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D019281:Dimerization; D006801:Humans; D007668:Kidney; D008247:Lysosomes / Q000201:enzymology; D009097:Multienzyme Complexes / Q000378:metabolism*; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D050722:Oncogene Protein v-cbl; D011480:Protease Inhibitors / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D025941:Protein Interaction Mapping; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D015682:Retroviridae Proteins, Oncogenic / Q000378:metabolism; D014162:Transfection; D025801:Ubiquitin / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D009097:Multienzyme Complexes; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D050722:Oncogene Protein v-cbl; D011480:Protease Inhibitors; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D015682:Retroviridae Proteins, Oncogenic; C433810:SH3KBP1 protein, human; C411889:Sh3kbp1 protein, rat; D025801:Ubiquitin; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex