The subcellular localization of 3-phosphoinositide-dependent protein kinase is controlled by caveolin-1 binding.
3-Phosphoinositide-dependent protein kinase 1 (PDK1), a member of the serine/threonine kinase family, has been demonstrated to be crucial for cellular survival, differentiation, and metabolism. Here, we present evidence that PDK1 is associated with caveolin-1, a 22-kDa integral membrane protein, which is the principal structural and regulatory component of the caveolae membranes in COS-1. First, we noted the presence of two potential caveolin-1 binding motifs ((141)FFVKLYFTF(149) and (299)YDFPEKFF(306)) in the PDK1 catalytic domain. Using a pull-down approach, we observed that PDK1 interacts physically with caveolin-1 both in vivo and in vitro. Second, we detected the co-localization of PDK1 and caveolin-1 via confocal microscopy. The localization of PDK1 to the plasma membrane was disrupted by caveolin binding. Third, in transient transfection assays, interaction with caveolin-1 induced a substantial reduction in the in vivo serine/threonine phosphorylation of PDK1, whereas the caveolin-1 binding site mutant ((141)FFVKLYFTF(149) and (299)YDFPEKFF(306) change to (141)AFVKLAFTA(149) and (299)ADAPEFLA(306)) did not. Furthermore, a caveolin-1 scaffolding peptide (amino acids 82-101) functionally suppressed the self-phosphorylation and kinase activities of purified recombinant PDK1 protein. Thus, our observations indicated that PDK1 binds to caveolin-1 through its caveolin-binding motifs, and also that the protein-protein interaction between PDK1 and caveolin-1 regulates PDK1 self-phosphorylation, kinase activity, and subcellular localization.
Chun
Jaesun
J
School of Science Education, Chungbuk National University, Chongju 361-763, Republic of Korea.
Kwon
Taegun
T
Lee
Eun Jeoung
EJ
Hyun
Sunghee
S
Hong
Soon-Kwang
SK
Kang
Sang Sun
SS
D064413:3-Phosphoinositide-Dependent Protein Kinases; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D051242:Caveolin 1; D022461:Caveolins / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D004789:Enzyme Activation; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D013347:Subcellular Fractions / Q000378:metabolism*; D013379:Substrate Specificity
C495540:CAV1 protein, human; D051242:Caveolin 1; D022461:Caveolins; D064413:3-Phosphoinositide-Dependent Protein Kinases; C578562:PDPK1 protein, human; D017346:Protein Serine-Threonine Kinases