Trihydrophobin 1 Interacts with PAK1 and Regulates ERK/MAPK Activation and Cell Migration.
The Rac1/Cdc42 effector, p21-activated kinase (PAK), is activated by various signaling cascades, including receptor-tyrosine kinases and integrins, and regulates a number of processes such as cell proliferation and motility. PAK activity has been shown to be required for maximal activation of the canonical RAF-MEK-MAPK signaling cascade, possibly because of PAK co-activation of RAF and MEK. Here we have shown that trihydrophobin 1 (TH1), originally identified as a negative regulator of A-RAF kinase, also interacted with PAK1 in cultured cells. Confocal microscopy assay indicated that TH1 colocalized with PAK1 in both the cytoplasm and nucleus, which is consistent with our previous results. GST pulldown and coimmunoprecipitation experiments demonstrated that TH1 interacted directly with PAK1 and bound selectively to the carboxyl-terminal kinase domain of PAK1, and the ability of the binding was enhanced along with activation of PAK1. The binding pattern of PAK1 implies that this interaction was mediated in part by PAK1 kinase activity. As indicated by in vitro kinase activity assays and Western blot detections, TH1 inhibited PAK1 kinase activity and negatively regulated MAPK signal transduction. Interestingly, TH1 bound with MEK1/ERK in cells and in vitro without directly suppressing their kinase activity. Furthermore, we observed that TH1 localized to focal adhesions and filopodia in the leading edge of cells, where TH1 reduced cell migration through affecting actin and adhesion dynamics. Based on these observations, we propose a model in which TH1 interacts with PAK1 and specifically restricts the activation of MAPK modules through the upstream region of the MAPK pathway, thereby influencing cell migration.
Cheng
Chunming
C
Gene Research Center, Shanghai Medical College, and Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, China.
Kong
Xiangfei
X
Wang
Hanzhou
H
Gan
Huachen
H
Hao
Yuqing
Y
Zou
Weiying
W
Wu
Jingwen
J
Chi
Yayun
Y
Yang
Junwu
J
Hong
Yi
Y
Chen
Kangli
K
Gu
Jianxin
J
D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002465:Cell Movement / Q000502:physiology*; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation / Q000502:physiology; D022001:Focal Adhesions / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D048369:MAP Kinase Kinase 1 / Q000235:genetics / Q000378:metabolism*; D020935:MAP Kinase Signaling System / Q000502:physiology*; D051379:Mice; D041681:NIH 3T3 Cells; D011485:Protein Binding / Q000502:physiology; D011554:Pseudopodia / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors; D054462:p21-Activated Kinases / Q000235:genetics / Q000378:metabolism*; D048490:raf Kinases
D002352:Carrier Proteins; D014157:Transcription Factors; C118437:negative elongation factor; C515903:PAK1 protein, human; C515905:Pak1 protein, mouse; D054462:p21-Activated Kinases; D048490:raf Kinases; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human; C482127:Map2k1 protein, mouse