Regulation of 3-phosphoinositide-dependent protein kinase-1 (PDK1) by Src involves tyrosine phosphorylation of PDK1 and Src homology 2 domain binding.
3-Phosphoinositide-dependent protein kinase-1 (PDK1) appears to play a central regulatory role in many cell signalings between phosphoinositide-3 kinase and various intracellular serine/threonine kinases. In resting cells, PDK1 is known to be constitutively active and is further activated by tyrosine phosphorylation (Tyr(9) and Tyr(373/376)) following the treatment of the cell with insulin or pervanadate. However, little is known about the mechanisms for this additional activation of PDK1. Here, we report that the SH2 domain of Src, Crk, and GAP recognized tyrosine-phosphorylated PDK1 in vitro. Destabilization of PDK1 induced by geldanamycin (a Hsp90 inhibitor) was partially blocked in HEK 293 cells expressing PDK1-Y9F. Co-expression of Hsp90 enhanced PDK1-Src complex formation and led to further increased PDK1 activity toward PKB and SGK. Immunohistochemical analysis with anti-phospho-Tyr(9) antibodies showed that the level of Tyr(9) phosphorylation was markedly increased in tumor samples compared with normal. Taken together, these data suggest that phosphorylation of PDK1 on Tyr(9), distinct from Tyr(373/376), is important for PDK1/Src complex formation, leading to PDK1 activation. Furthermore, Tyr(9) phosphorylation is critical for the stabilization of both PDK1 and the PDK1/Src complex via Hsp90-mediated protection of PDK1 degradation.
Yang
Keum-Jin
KJ
Department of Pharmacology, Daejeon Regional Cancer Center, Cancer Research Institute, Research Institute for Medical Sciences, Taejeon 301-131, South Korea.
Shin
Sanghee
S
Department of Pharmacology, Daejeon Regional Cancer Center, Cancer Research Institute, Research Institute for Medical Sciences, Taejeon 301-131, South Korea.
Piao
Longzhen
L
Department of Pharmacology, Daejeon Regional Cancer Center, Cancer Research Institute, Research Institute for Medical Sciences, Taejeon 301-131, South Korea.
Shin
Eulsoon
E
Department of Pharmacology, Daejeon Regional Cancer Center, Cancer Research Institute, Research Institute for Medical Sciences, Taejeon 301-131, South Korea.
Li
Yuwen
Y
Department of Pharmacology, Daejeon Regional Cancer Center, Cancer Research Institute, Research Institute for Medical Sciences, Taejeon 301-131, South Korea.
Park
Kyeong Ah
KA
Department of Pharmacology, Daejeon Regional Cancer Center, Cancer Research Institute, Research Institute for Medical Sciences, Taejeon 301-131, South Korea.
Byun
Hee Sun
HS
Department of Pharmacology, Daejeon Regional Cancer Center, Cancer Research Institute, Research Institute for Medical Sciences, Taejeon 301-131, South Korea.
Won
Minho
M
Department of Pharmacology, Daejeon Regional Cancer Center, Cancer Research Institute, Research Institute for Medical Sciences, Taejeon 301-131, South Korea.
Hong
Janghee
J
Department of Pharmacology, Daejeon Regional Cancer Center, Cancer Research Institute, Research Institute for Medical Sciences, Taejeon 301-131, South Korea.
Kweon
Gi Ryang
GR
Department of Biochemistry, College of Medicine, Chungnam National University, Taejeon 301-131, South Korea.
Hur
Gang Min
GM
Department of Pharmacology, Daejeon Regional Cancer Center, Cancer Research Institute, Research Institute for Medical Sciences, Taejeon 301-131, South Korea.
Seok
Jeong Ho
JH
Department of Pharmacology, Daejeon Regional Cancer Center, Cancer Research Institute, Research Institute for Medical Sciences, Taejeon 301-131, South Korea.
Chun
Taehoon
T
Division of Biotechnology, School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, South Korea.
Brazil
Derek P
DP
University College Dublin School of Biomolecular and Biomedical Science, University College Dublin Conway Institute, University College Dublin, Dublin 4, Ireland.
Hemmings
Brian A
BA
Friedrich Miescher Institute for Biomedical Research, Basel CH-4058, Switzerland.
Park
Jongsun
J
Department of Pharmacology, Daejeon Regional Cancer Center, Cancer Research Institute, Research Institute for Medical Sciences, Taejeon 301-131, South Korea. Electronic address: insulin@cnu.ac.kr.
D064413:3-Phosphoinositide-Dependent Protein Kinases; D002460:Cell Line; D004194:Disease; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability / Q000187:drug effects; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D008954:Models, Biological; D050505:Mutant Proteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000378:metabolism*; D061988:Proteasome Inhibitors; D011485:Protein Binding / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects; D050737:Proto-Oncogene Proteins c-crk / Q000378:metabolism; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000737:chemistry* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000201:enzymology; D018909:src Homology Domains
D018841:HSP90 Heat-Shock Proteins; D007976:Leupeptins; D050505:Mutant Proteins; D061988:Proteasome Inhibitors; D050737:Proto-Oncogene Proteins c-crk; D011993:Recombinant Fusion Proteins; D019000:Phosphotyrosine; D016392:Proto-Oncogene Proteins pp60(c-src); D064413:3-Phosphoinositide-Dependent Protein Kinases; C578562:PDPK1 protein, human; D017346:Protein Serine-Threonine Kinases; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde