Abstract
PAC-1 is an inducible, nuclear-specific, dual-specificity mitogen-activated protein (MAP) kinase phosphatase that has been shown recently to be a transcription target of the human tumor-suppressor protein p53 in signaling apoptosis and growth suppression. However, its substrate specificity and regulation of catalytic activity thus far remain elusive. Here, we report in vitro characterization of PAC-1 phosphatase activity with three distinct MAP kinase subfamilies. We show that the recombinant PAC-1 exists in a virtually inactive state when alone in vitro, and dephosphorylates extracellular signal-regulated kinase 2 (ERK2) but not p38α or c-Jun NH 2-terminal kinase 2 (JNK2). ERK2 dephosphorylation by PAC-1 requires association of its amino-terminal domain with ERK2 that results in catalytic activation of the phosphatase. p38α also interacts with but does not activate PAC-1, whereas JNK2 does not bind to or cause catalytic activation by PAC-1. Moreover, our structure-based analysis reveals that individual mutation of the conserved Arg294 and Arg295 that likely comprise the phosphothreonine- binding pocket in PAC-1 to either alanine or lysine results in a nearly complete loss of its phosphatase activity even in the presence of ERK2. These results suggest that Arg294 and Arg295 play an important role in PAC-1 catalytic activation induced by ERK2 binding.
| Original language | English |
|---|---|
| Pages (from-to) | 777-788 |
| Number of pages | 12 |
| Journal | Journal of Molecular Biology |
| Volume | 354 |
| Issue number | 4 |
| DOIs | |
| State | Published - Dec 9 2005 |
| Externally published | Yes |
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Keywords
- c-Jun NH-terminal kinase
- Extracellular signal-regulated kinase
- MAPK phosphatase
- Mitogen-activated protein kinase
- PAC-1
ASJC Scopus subject areas
- Virology
Cite this
New insights into the catalytic activation of the MAPK phosphatase PAC-1 induced by its substrate MAPK ERK2 binding. / Zhang, Qiang; Muller, Michaela; Chen, Can Hao; Zeng, Lei; Farooq, Amjad; Zhou, Ming Ming.
In: Journal of Molecular Biology, Vol. 354, No. 4, 09.12.2005, p. 777-788.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - New insights into the catalytic activation of the MAPK phosphatase PAC-1 induced by its substrate MAPK ERK2 binding
AU - Zhang, Qiang
AU - Muller, Michaela
AU - Chen, Can Hao
AU - Zeng, Lei
AU - Farooq, Amjad
AU - Zhou, Ming Ming
PY - 2005/12/9
Y1 - 2005/12/9
N2 - PAC-1 is an inducible, nuclear-specific, dual-specificity mitogen-activated protein (MAP) kinase phosphatase that has been shown recently to be a transcription target of the human tumor-suppressor protein p53 in signaling apoptosis and growth suppression. However, its substrate specificity and regulation of catalytic activity thus far remain elusive. Here, we report in vitro characterization of PAC-1 phosphatase activity with three distinct MAP kinase subfamilies. We show that the recombinant PAC-1 exists in a virtually inactive state when alone in vitro, and dephosphorylates extracellular signal-regulated kinase 2 (ERK2) but not p38α or c-Jun NH 2-terminal kinase 2 (JNK2). ERK2 dephosphorylation by PAC-1 requires association of its amino-terminal domain with ERK2 that results in catalytic activation of the phosphatase. p38α also interacts with but does not activate PAC-1, whereas JNK2 does not bind to or cause catalytic activation by PAC-1. Moreover, our structure-based analysis reveals that individual mutation of the conserved Arg294 and Arg295 that likely comprise the phosphothreonine- binding pocket in PAC-1 to either alanine or lysine results in a nearly complete loss of its phosphatase activity even in the presence of ERK2. These results suggest that Arg294 and Arg295 play an important role in PAC-1 catalytic activation induced by ERK2 binding.
AB - PAC-1 is an inducible, nuclear-specific, dual-specificity mitogen-activated protein (MAP) kinase phosphatase that has been shown recently to be a transcription target of the human tumor-suppressor protein p53 in signaling apoptosis and growth suppression. However, its substrate specificity and regulation of catalytic activity thus far remain elusive. Here, we report in vitro characterization of PAC-1 phosphatase activity with three distinct MAP kinase subfamilies. We show that the recombinant PAC-1 exists in a virtually inactive state when alone in vitro, and dephosphorylates extracellular signal-regulated kinase 2 (ERK2) but not p38α or c-Jun NH 2-terminal kinase 2 (JNK2). ERK2 dephosphorylation by PAC-1 requires association of its amino-terminal domain with ERK2 that results in catalytic activation of the phosphatase. p38α also interacts with but does not activate PAC-1, whereas JNK2 does not bind to or cause catalytic activation by PAC-1. Moreover, our structure-based analysis reveals that individual mutation of the conserved Arg294 and Arg295 that likely comprise the phosphothreonine- binding pocket in PAC-1 to either alanine or lysine results in a nearly complete loss of its phosphatase activity even in the presence of ERK2. These results suggest that Arg294 and Arg295 play an important role in PAC-1 catalytic activation induced by ERK2 binding.
KW - c-Jun NH-terminal kinase
KW - Extracellular signal-regulated kinase
KW - MAPK phosphatase
KW - Mitogen-activated protein kinase
KW - PAC-1
UR - http://www.scopus.com/inward/record.url?scp=28444434759&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=28444434759&partnerID=8YFLogxK
U2 - 10.1016/j.jmb.2005.10.006
DO - 10.1016/j.jmb.2005.10.006
M3 - Article
C2 - 16288922
AN - SCOPUS:28444434759
VL - 354
SP - 777
EP - 788
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
SN - 0022-2836
IS - 4
ER -