Protein phosphatase-1 modulates the function of Pax-6, a transcription factor controlling brain and eye development.
Pax-6 is an evolutionarily conserved transcription factor and acts high up in the regulatory hierarchy controlling eye and brain development in humans, mice, zebrafish, and Drosophila. Previous studies have shown that Pax-6 is a phosphoprotein, and its phosphorylation by ERK, p38, and homeodomain-interacting protein kinase 2 greatly enhances its transactivation activity. However, the protein phosphatases responsible for the dephosphorylation of Pax-6 remain unknown. Here, we present both in vitro and in vivo evidence to show that protein serine/threonine phosphatase-1 is a major phosphatase that directly dephosphorylates Pax-6. First, purified protein phosphatase-1 directly dephosphorylates Pax-6 in vitro. Second, immunoprecipitation-linked Western blot revealed that both protein phosphatase-1alpha and protein phosphatase-1beta interact with Pax-6. Third, overexpression of protein phosphatase-1 in human lens epithelial cells leads to dephosphorylation of Pax-6. Finally, inhibition of protein phosphatase-1 activity by calyculin A or knockdown of protein phosphatase-1alpha and protein phosphatase-1beta by RNA interference leads to enhanced phosphorylation of Pax-6. Moreover, our results also demonstrate that dephosphorylation of Pax-6 by protein phosphatase-1 significantly modulates its function in regulating expression of both exogenous and endogenous genes. These results demonstrate that protein phosphatase 1 acts as a major phosphatase to dephosphorylate Pax-6 and modulate its function.
Yan
Qin
Q
Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68198, USA.
Liu
Wen-Bin
WB
Qin
Jichao
J
Liu
Jinping
J
Chen
He-Ge
HG
Huang
Xiaoqin
X
Chen
Lili
L
Sun
Shuming
S
Deng
Mi
M
Gong
Lili
L
Li
Yong
Y
Zhang
Lan
L
Liu
Yan
Y
Feng
Hao
H
Xiao
Yamei
Y
Liu
Yun
Y
Li
David W-C
DW
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000254:growth & development*; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology; D005123:Eye / Q000254:growth & development*; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007908:Lens, Crystalline / Q000187:drug effects* / Q000201:enzymology; D008387:Marine Toxins; D051379:Mice; D008969:Molecular Sequence Data; D010080:Oxazoles / Q000494:pharmacology; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors / Q000235:genetics / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D010766:Phosphorylation; D011485:Protein Binding; D054645:Protein Phosphatase 1; D034741:RNA, Small Interfering / Q000494:pharmacology; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic
D004791:Enzyme Inhibitors; D005136:Eye Proteins; D018398:Homeodomain Proteins; D008387:Marine Toxins; D010080:Oxazoles; D000071841:PAX6 Transcription Factor; C000605730:PAX6 protein, human; D051761:Paired Box Transcription Factors; C000605732:Pax6 protein, mouse; D034741:RNA, Small Interfering; D012097:Repressor Proteins; C059041:calyculin A; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1