Abstract
Phosphatidylethanolamine binding proteins (PEBP) represent a superfamily of proteins that are conserved from bacteria to humans. In mammals, four members have been identified, PEBP1-4. To determine the functional differences among PEBP1-4 and the underlying mechanism for their actions, we performed a sequence alignment and found that PEBP4 contains a signal peptide and potential glycosylation sites, whereas PEBP1-3 are intracellular proteins. To test if PEBP4 is secreted, we made constructs with Myc epitope at the amino (N) terminus or carboxyl (C) terminus to mask the signal sequence or keep it free, respectively. Our data revealed that both mouse and human PEBP4 were secreted when the epitope was tagged at their C-terminus. To our surprise, secretion was dependent upon the C-terminal conserved domain in addition to the N-terminal signal sequence. When the epitope was placed to the N-terminus, the recombinant protein failed to secrete and instead, was retained in the cytoplasm. Mass spectrometry detected asparagine (N)-glycosylation on the secreted PEBP4. Although overexpression of N-terminal tagged PEBP4 resulted in an inhibition of ERK activation by EGF, that with a C-terminal epitope tag did not have such an effect. Likewise, transfection of PEBP4 shRNA did not appear to affect ERK activation, suggesting that PEBP4 does not participate in the regulation of this pathway. In contrast, PEBP4 siRNA suppressed phosphorylation of Act at S473. Therefore, our results suggest that PEBP4 is a multifunctional protein and can be secreted. It will be important to investigate the mechanism by which PEBP4 is secreted and regulates cellular events.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 1682-1689 |
| Number of pages | 8 |
| Journal | Biochimica et Biophysica Acta - Molecular Cell Research |
| Volume | 1863 |
| Issue number | 7 |
| DOIs | |
| State | Published - Jul 1 2016 |
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Keywords
- Act
- ERK
- Glycosylation
- PEBP4
- Secretion
- Signal peptide
ASJC Scopus subject areas
- Cell Biology
- Molecular Biology
Cite this
Phosphatidylethanolamine binding protein 4 (PEBP4) is a secreted protein and has multiple functions. / He, Huan; Liu, Dan; Lin, Hui; Jiang, Shanshan; Ying, Ying; Chun, Shao; Deng, Haiteng; Zaia, Joseph; Wen, Rong; Luo, Zhijun.
In: Biochimica et Biophysica Acta - Molecular Cell Research, Vol. 1863, No. 7, 01.07.2016, p. 1682-1689.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Phosphatidylethanolamine binding protein 4 (PEBP4) is a secreted protein and has multiple functions
AU - He, Huan
AU - Liu, Dan
AU - Lin, Hui
AU - Jiang, Shanshan
AU - Ying, Ying
AU - Chun, Shao
AU - Deng, Haiteng
AU - Zaia, Joseph
AU - Wen, Rong
AU - Luo, Zhijun
PY - 2016/7/1
Y1 - 2016/7/1
N2 - Phosphatidylethanolamine binding proteins (PEBP) represent a superfamily of proteins that are conserved from bacteria to humans. In mammals, four members have been identified, PEBP1-4. To determine the functional differences among PEBP1-4 and the underlying mechanism for their actions, we performed a sequence alignment and found that PEBP4 contains a signal peptide and potential glycosylation sites, whereas PEBP1-3 are intracellular proteins. To test if PEBP4 is secreted, we made constructs with Myc epitope at the amino (N) terminus or carboxyl (C) terminus to mask the signal sequence or keep it free, respectively. Our data revealed that both mouse and human PEBP4 were secreted when the epitope was tagged at their C-terminus. To our surprise, secretion was dependent upon the C-terminal conserved domain in addition to the N-terminal signal sequence. When the epitope was placed to the N-terminus, the recombinant protein failed to secrete and instead, was retained in the cytoplasm. Mass spectrometry detected asparagine (N)-glycosylation on the secreted PEBP4. Although overexpression of N-terminal tagged PEBP4 resulted in an inhibition of ERK activation by EGF, that with a C-terminal epitope tag did not have such an effect. Likewise, transfection of PEBP4 shRNA did not appear to affect ERK activation, suggesting that PEBP4 does not participate in the regulation of this pathway. In contrast, PEBP4 siRNA suppressed phosphorylation of Act at S473. Therefore, our results suggest that PEBP4 is a multifunctional protein and can be secreted. It will be important to investigate the mechanism by which PEBP4 is secreted and regulates cellular events.
AB - Phosphatidylethanolamine binding proteins (PEBP) represent a superfamily of proteins that are conserved from bacteria to humans. In mammals, four members have been identified, PEBP1-4. To determine the functional differences among PEBP1-4 and the underlying mechanism for their actions, we performed a sequence alignment and found that PEBP4 contains a signal peptide and potential glycosylation sites, whereas PEBP1-3 are intracellular proteins. To test if PEBP4 is secreted, we made constructs with Myc epitope at the amino (N) terminus or carboxyl (C) terminus to mask the signal sequence or keep it free, respectively. Our data revealed that both mouse and human PEBP4 were secreted when the epitope was tagged at their C-terminus. To our surprise, secretion was dependent upon the C-terminal conserved domain in addition to the N-terminal signal sequence. When the epitope was placed to the N-terminus, the recombinant protein failed to secrete and instead, was retained in the cytoplasm. Mass spectrometry detected asparagine (N)-glycosylation on the secreted PEBP4. Although overexpression of N-terminal tagged PEBP4 resulted in an inhibition of ERK activation by EGF, that with a C-terminal epitope tag did not have such an effect. Likewise, transfection of PEBP4 shRNA did not appear to affect ERK activation, suggesting that PEBP4 does not participate in the regulation of this pathway. In contrast, PEBP4 siRNA suppressed phosphorylation of Act at S473. Therefore, our results suggest that PEBP4 is a multifunctional protein and can be secreted. It will be important to investigate the mechanism by which PEBP4 is secreted and regulates cellular events.
KW - Act
KW - ERK
KW - Glycosylation
KW - PEBP4
KW - Secretion
KW - Signal peptide
UR - http://www.scopus.com/inward/record.url?scp=84964906899&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84964906899&partnerID=8YFLogxK
U2 - 10.1016/j.bbamcr.2016.03.022
DO - 10.1016/j.bbamcr.2016.03.022
M3 - Article
C2 - 27033522
AN - SCOPUS:84964906899
VL - 1863
SP - 1682
EP - 1689
JO - Biochimica et Biophysica Acta - Molecular Cell Research
JF - Biochimica et Biophysica Acta - Molecular Cell Research
SN - 0167-4889
IS - 7
ER -