The CCT/TRiC chaperonin is required for maturation of sphingosine kinase 1.
Sphingosine kinase 1 (SK1) catalyses the generation of sphingosine 1-phosphate (S1P), a bioactive phospholipid that influences a diverse range of cellular processes, including proliferation, survival, adhesion, migration, morphogenesis and differentiation. SK1 is controlled by various mechanisms, including transcriptional regulation, and post-translational activation by phosphorylation and protein-protein interactions which can regulate both the activity and localisation of this enzyme. To gain a better understanding of the regulatory mechanisms controlling SK1 activity and function we performed a yeast two-hybrid screen to identify SK1-interacting proteins. Using this approach we identified that SK1 interacts with subunit 7 (eta) of cytosolic chaperonin CCT (chaperonin containing t-complex polypeptide, also called TRiC for TCP-1 ring complex), a hexadecameric chaperonin that binds unfolded polypeptides and mediates their folding and release in an ATP-dependent manner. Further analysis of the SK1-CCTeta interaction demonstrated that other CCT/TRiC subunits also associated with SK1 in HEK293T cell lysates in an ATP-sensitive manner, suggesting that the intact, functional, multimeric CCT/TRiC complex associated with SK1. Furthermore, pulse-chase studies indicated that CCT/TRiC binds specifically to newly translated SK1. Finally, depletion of functional CCT/TRiC through the use of RNA interference in HeLa cells or temperature sensitive CCT yeast mutants reduced cellular SK1 activity. Thus, combined this data suggests that SK1 is a CCT/TRiC substrate, and that this chaperonin facilitates folding of newly translated SK1 into its mature active form.
Zebol
Julia R
JR
Hanson Institute, Division of Human Immunology, Institute of Medical and Veterinary Science, Frome Road, Adelaide, SA 5000, Australia.
Hewitt
Niamh M
NM
Moretti
Paul A B
PA
Lynn
Helen E
HE
Lake
Julie A
JA
Li
Peng
P
Vadas
Mathew A
MA
Wattenberg
Binks W
BW
Pitson
Stuart M
SM
D056404:Chaperonin Containing TCP-1; D018833:Chaperonins / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007962:Leukocytes / Q000378:metabolism; D008246:Lysophospholipids / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D017510:Protein Folding; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D013110:Sphingosine / Q000031:analogs & derivatives / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection; D020798:Two-Hybrid System Techniques; D000099157:Sphingosine Kinase
D056404:Chaperonin Containing TCP-1; D018833:Chaperonins; D008246:Lysophospholipids; D017853:Phosphotransferases (Alcohol Group Acceptor); D013110:Sphingosine; D000099157:Sphingosine Kinase; C060506:sphingosine 1-phosphate