Plk3 functions as an essential component of the hypoxia regulatory pathway by direct phosphorylation of HIF-1alpha.
Polo-like kinase 3 (Plk3) plays an important role in the regulation of cell cycle progression and stress responses. Plk3 also has a tumor-suppressing activity as aging PLK3-null mice develop tumors in multiple organs. The growth of highly vascularized tumors in PLK3-null mice suggests a role for Plk3 in angiogenesis and cellular responses to hypoxia. By studying primary isogenic murine embryonic fibroblasts, we tested the hypothesis that Plk3 functions as a component in the hypoxia signaling pathway. PLK3(-/-) murine embryonic fibroblasts contained an enhanced level of HIF-1α under hypoxic conditions. Immunoprecipitation and pulldown analyses revealed that Plk3 physically interacted with HIF-1α under hypoxia. Purified recombinant Plk3, but not a kinase-defective mutant, phosphorylated HIF-1α in vitro, resulting in a major mobility shift. Mass spectrometry identified two unique serine residues that were phosphorylated by Plk3. Moreover, ectopic expression followed by cycloheximide or pulse-chase treatment demonstrated that phospho-mutants exhibited a much longer half-life than the wild-type counterpart, strongly suggesting that Plk3 directly regulates HIF-1α stability in vivo. Combined, our study identifies Plk3 as a new and essential player in the regulation of the hypoxia signaling pathway.
Xu
Dazhong
D
Department of Environmental Medicine and Pharmacology, New York University School of Medicine, Tuxedo, New York 10987, USA.
Yao
Yixin
Y
Lu
Luo
L
Costa
Max
M
Dai
Wei
W
D000818:Animals; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002470:Cell Survival; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D000860:Hypoxia; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry; D015398:Signal Transduction; D025521:Tumor Suppressor Proteins; D000097868:Polo-like Kinases
D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D017346:Protein Serine-Threonine Kinases; D011994:Recombinant Proteins; D025521:Tumor Suppressor Proteins; C486256:PLK3 protein, human; D000097868:Polo-like Kinases; C093328:Plk3 protein, mouse