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Label-free quantitative phosphorylation analysis of human transgelin2 in Jurkat T cells reveals distinct phosphorylation patterns under PKA and PKC activation conditions.

Jang SH, Jun CD, Park ZY - Proteome Sci (2015)

Bottom Line: Most phosphorylation sites showing specific kinase-dependent phosphorylation changes were discretely located in two previously characterized actin-binding regions: actin-binding site (ABS) and calponin repeat domain (CNR).PKC activation increased phosphorylation of threonine-180 and serine-185 in the CNR, and PKA activation increased phosphorylation of serine-163 in the ABS.Multiple actin-binding regions of transgelin2 participate to accomplish its full actin-binding capability, and the actin-binding affinity of each actin-binding region appears to be modulated by specific kinase-dependent phosphorylation changes.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Gwangju Institute of Science & Technology, 123, Cheomdangwagi-Ro, Buk-Gu, 500-712 Gwangju Republic of Korea.

ABSTRACT

Background: Transgelin2, one of cytoskeletal actin binding proteins has recently been suggested to be involved in the formation of immune synapses. Although detailed function of transgelin2 is largely unknown, interactions between transgelin2 and actin appear to be important in regulating cellular functions of transgelin2. Because protein phosphorylation can change ability to interact with other proteins, comprehensive phosphorylation analysis of transgelin2 will be helpful in understanding its functional mechanisms.

Results: Here, a specific protein label-free quantitative phosphorylation analysis method combining immuno-precipitation, IMAC phosphopeptide enrichment technique and label-free relative quantification analysis was used to monitor the phosphorylation changes of transgelin2 overexpressed in Jurkat T cells under protein kinase C (PKC) and protein kinase A (PKA) activation conditions, two representative intracellular signalling pathways of immune cell activation and homeostasis. A total of six serine/threonine phosphorylation sites were identified including threonine-84, a novel phosphorylation site. Notably, distinct phosphorylation patterns of transgelin2 under the two kinase activation conditions were observed. Most phosphorylation sites showing specific kinase-dependent phosphorylation changes were discretely located in two previously characterized actin-binding regions: actin-binding site (ABS) and calponin repeat domain (CNR). PKC activation increased phosphorylation of threonine-180 and serine-185 in the CNR, and PKA activation increased phosphorylation of serine-163 in the ABS.

Conclusions: Multiple actin-binding regions of transgelin2 participate to accomplish its full actin-binding capability, and the actin-binding affinity of each actin-binding region appears to be modulated by specific kinase-dependent phosphorylation changes. Accordingly, different actin-binding properties or cellular functions of transgelin2 may result from distinct intracellular signalling events under immune response activation or homeostasis conditions.

No MeSH data available.


Related in: MedlinePlus

Calyculin A treatment effects in the phosphorylation analysis of transgelin2. ProQ-Diamond and Coomassie sequential staining results of GFP tagged transgelin2 following SDS-PAGE separation (A and B), and micro RPLC-MS/MS analysis results (C).
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Fig1: Calyculin A treatment effects in the phosphorylation analysis of transgelin2. ProQ-Diamond and Coomassie sequential staining results of GFP tagged transgelin2 following SDS-PAGE separation (A and B), and micro RPLC-MS/MS analysis results (C).

Mentions: Conventional phosphatase inhibitor treatments of the cell lysates such as phosphatase inhibitor cocktails did not effectively improve the identification of transgelin2 Ser/Thr phosphorylation sites. Only one or two phosphorylation sites were typically identified. To increase the number of transgelin2 phosphorylation sites identified in the quantitative phosphorylation analysis, various phosphatase inhibitors such as calyculin A and okadaic acid were tested. Among the phosphatase inhibitors tested in our laboratory, calyculin A treatment exhibited the most significant changes in terms of the total numbers of identified phosphorylation sites and phosphopeptides. To show the phosphatase inhibitory effects of calyculin A, Jurkat T cells overexpressing GFP-tagged transgelin2 were treated with calyculin A for 30 min, okadaic acid for 30 min or untreated prior to cell harvest. These three cell samples were lysed separately with lysis buffers containing conventional phosphatase inhibitors, followed by immuno-precipitations with anti-GFP antibodies. The immuno-purified GFP-tagged transgelin2 samples were then subjected to SDS-PAGE and the resulting SDS-PAGE gel was sequentially stained with Pro-Q Diamond and Coomassie. The Coomassie staining results showed one major protein band that matched the molecular weight of GFP-tagged transgelin2, and the intensities of this protein band were not noticeably different among the untreated and two phosphatase-treated conditions (Figure 1A). In contrast, the Pro-Q Diamond staining results indicated significantly higher phosphorylation levels of transgelin2 in calyculin A-treated samples (Figure 1B). The increased phosphorylation level of the GFP-tagged transgelin2 in calyculin A-treated sample was further confirmed by micro RPLC-MS/MS analysis. The number of unique phosphopeptide identified was increased from 4 to 9 and a total of 6 phosphorylation sites were identified in the samples with the calyculin A treatment. The total number of MS/MS spectra assigned to phosphopeptides was increased by more than four-fold from 12 to 56 through the use of the calyculin A treatment (Figure 1C). Among the phosphorylation sites identified in the calyculin A-treated sample, threonine-84 has not been previously identified by mass spectrometry. MS/MS spectrum of the phosphopeptide containing threonine-84 was manually validated, and the results are shown in Additional file 1: Figure S1.Figure 1


Label-free quantitative phosphorylation analysis of human transgelin2 in Jurkat T cells reveals distinct phosphorylation patterns under PKA and PKC activation conditions.

Jang SH, Jun CD, Park ZY - Proteome Sci (2015)

Calyculin A treatment effects in the phosphorylation analysis of transgelin2. ProQ-Diamond and Coomassie sequential staining results of GFP tagged transgelin2 following SDS-PAGE separation (A and B), and micro RPLC-MS/MS analysis results (C).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4384351&req=5

Fig1: Calyculin A treatment effects in the phosphorylation analysis of transgelin2. ProQ-Diamond and Coomassie sequential staining results of GFP tagged transgelin2 following SDS-PAGE separation (A and B), and micro RPLC-MS/MS analysis results (C).
Mentions: Conventional phosphatase inhibitor treatments of the cell lysates such as phosphatase inhibitor cocktails did not effectively improve the identification of transgelin2 Ser/Thr phosphorylation sites. Only one or two phosphorylation sites were typically identified. To increase the number of transgelin2 phosphorylation sites identified in the quantitative phosphorylation analysis, various phosphatase inhibitors such as calyculin A and okadaic acid were tested. Among the phosphatase inhibitors tested in our laboratory, calyculin A treatment exhibited the most significant changes in terms of the total numbers of identified phosphorylation sites and phosphopeptides. To show the phosphatase inhibitory effects of calyculin A, Jurkat T cells overexpressing GFP-tagged transgelin2 were treated with calyculin A for 30 min, okadaic acid for 30 min or untreated prior to cell harvest. These three cell samples were lysed separately with lysis buffers containing conventional phosphatase inhibitors, followed by immuno-precipitations with anti-GFP antibodies. The immuno-purified GFP-tagged transgelin2 samples were then subjected to SDS-PAGE and the resulting SDS-PAGE gel was sequentially stained with Pro-Q Diamond and Coomassie. The Coomassie staining results showed one major protein band that matched the molecular weight of GFP-tagged transgelin2, and the intensities of this protein band were not noticeably different among the untreated and two phosphatase-treated conditions (Figure 1A). In contrast, the Pro-Q Diamond staining results indicated significantly higher phosphorylation levels of transgelin2 in calyculin A-treated samples (Figure 1B). The increased phosphorylation level of the GFP-tagged transgelin2 in calyculin A-treated sample was further confirmed by micro RPLC-MS/MS analysis. The number of unique phosphopeptide identified was increased from 4 to 9 and a total of 6 phosphorylation sites were identified in the samples with the calyculin A treatment. The total number of MS/MS spectra assigned to phosphopeptides was increased by more than four-fold from 12 to 56 through the use of the calyculin A treatment (Figure 1C). Among the phosphorylation sites identified in the calyculin A-treated sample, threonine-84 has not been previously identified by mass spectrometry. MS/MS spectrum of the phosphopeptide containing threonine-84 was manually validated, and the results are shown in Additional file 1: Figure S1.Figure 1

Bottom Line: Most phosphorylation sites showing specific kinase-dependent phosphorylation changes were discretely located in two previously characterized actin-binding regions: actin-binding site (ABS) and calponin repeat domain (CNR).PKC activation increased phosphorylation of threonine-180 and serine-185 in the CNR, and PKA activation increased phosphorylation of serine-163 in the ABS.Multiple actin-binding regions of transgelin2 participate to accomplish its full actin-binding capability, and the actin-binding affinity of each actin-binding region appears to be modulated by specific kinase-dependent phosphorylation changes.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Gwangju Institute of Science & Technology, 123, Cheomdangwagi-Ro, Buk-Gu, 500-712 Gwangju Republic of Korea.

ABSTRACT

Background: Transgelin2, one of cytoskeletal actin binding proteins has recently been suggested to be involved in the formation of immune synapses. Although detailed function of transgelin2 is largely unknown, interactions between transgelin2 and actin appear to be important in regulating cellular functions of transgelin2. Because protein phosphorylation can change ability to interact with other proteins, comprehensive phosphorylation analysis of transgelin2 will be helpful in understanding its functional mechanisms.

Results: Here, a specific protein label-free quantitative phosphorylation analysis method combining immuno-precipitation, IMAC phosphopeptide enrichment technique and label-free relative quantification analysis was used to monitor the phosphorylation changes of transgelin2 overexpressed in Jurkat T cells under protein kinase C (PKC) and protein kinase A (PKA) activation conditions, two representative intracellular signalling pathways of immune cell activation and homeostasis. A total of six serine/threonine phosphorylation sites were identified including threonine-84, a novel phosphorylation site. Notably, distinct phosphorylation patterns of transgelin2 under the two kinase activation conditions were observed. Most phosphorylation sites showing specific kinase-dependent phosphorylation changes were discretely located in two previously characterized actin-binding regions: actin-binding site (ABS) and calponin repeat domain (CNR). PKC activation increased phosphorylation of threonine-180 and serine-185 in the CNR, and PKA activation increased phosphorylation of serine-163 in the ABS.

Conclusions: Multiple actin-binding regions of transgelin2 participate to accomplish its full actin-binding capability, and the actin-binding affinity of each actin-binding region appears to be modulated by specific kinase-dependent phosphorylation changes. Accordingly, different actin-binding properties or cellular functions of transgelin2 may result from distinct intracellular signalling events under immune response activation or homeostasis conditions.

No MeSH data available.


Related in: MedlinePlus