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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

PKC-dependent phosphorylation changes of transgelin2 serine-185. Selected ion chromatograms of two isobaric phosphopeptides containing one oxidized methionine (183GApSQAGMoxTGYGMPR196 and 183GApSQAGMTGYGMoxPR196) (A and B) and the same sequence phosphopeptide with two oxidized methionines under no activation and PKC activation condition (C and D). * denotes 183GApSQAGMTGYGMoxPR196 and ** denotes 183GApSQAGMoxTGYGMPR196.
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Fig3: PKC-dependent phosphorylation changes of transgelin2 serine-185. Selected ion chromatograms of two isobaric phosphopeptides containing one oxidized methionine (183GApSQAGMoxTGYGMPR196 and 183GApSQAGMTGYGMoxPR196) (A and B) and the same sequence phosphopeptide with two oxidized methionines under no activation and PKC activation condition (C and D). * denotes 183GApSQAGMTGYGMoxPR196 and ** denotes 183GApSQAGMoxTGYGMPR196.

Mentions: Our label-free quantitative phosphorylation analysis method was then used to investigate the phosphorylation level changes of GFP-tagged transgelin2 overexpressed in Jurkat T cells under PKC activation conditions. The most significant phosphorylation level change was observed with serine-185, in which the phosphorylation increased by more than two-fold. A manually validated MS/MS spectrum and selected ion chromatograms of the phosphopeptide containing serine-185 with or without PMA treatment are shown in Figure 2. Other phosphorylation sites showing significant phosphorylation level changes included serine-11 and threonine-180 (Additional file 4: Figure S3 and Additional file 5: Figure S4). Additionally, phosphopeptides containing methionine residue(s) were identified as discrete phosphopeptides, depending on the numbers and locations of oxidized methionine. For example, serine-185 co-localized with two methionine residues in the same tryptic digest peptide was identified in four different phosphopeptides. Two isobaric phosphopeptides, 183GAS*QAGMoxTGYGMPR196 and 183GAS*QAGMTGYGMoxPR196, whose only difference consisted of the location of the oxidized methionine, were clearly separated in the RPLC separation and unambiguously identified (Figure 3A and B). Finally, selected ion chromatograms of the phosphopeptides containing serine-185 with two oxidized methionines are shown in Figure 3C and D. The phosphorylation of transgelin2 under the PKA activation condition was also investigated by our quantitative phosphorylation analysis method. Although the number of phosphorylation sites and phosphopeptides identified were identical compared with those from the PKC activation, a moderate level of phosphorylation change (~36% increase) was observed only at serine-163 (Additional file 6: Figure S5). To increase the confidence level of our transgelin2 quantitative phosphorylation analysis results, the PKA, PKC and no activation condition experiments were independently performed three times. The peak areas of the individual phosphopeptides from the three independent experiments under the above two kinase activation conditions were calculated and plotted in terms of the peak area ratios compared with the no activation condition experiments (Figure 4 and Additional file 7: Figure S6). The preferential phosphorylation sites of transgelin2 under the PKA and PKC activation conditions appeared to vary. Serine-11, threonine-180 and serine-185 were responsive solely to PKC activation, whereas serine-163 is responsive to PKA activation.Figure 2


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)

PKC-dependent phosphorylation changes of transgelin2 serine-185. Selected ion chromatograms of two isobaric phosphopeptides containing one oxidized methionine (183GApSQAGMoxTGYGMPR196 and 183GApSQAGMTGYGMoxPR196) (A and B) and the same sequence phosphopeptide with two oxidized methionines under no activation and PKC activation condition (C and D). * denotes 183GApSQAGMTGYGMoxPR196 and ** denotes 183GApSQAGMoxTGYGMPR196.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: PKC-dependent phosphorylation changes of transgelin2 serine-185. Selected ion chromatograms of two isobaric phosphopeptides containing one oxidized methionine (183GApSQAGMoxTGYGMPR196 and 183GApSQAGMTGYGMoxPR196) (A and B) and the same sequence phosphopeptide with two oxidized methionines under no activation and PKC activation condition (C and D). * denotes 183GApSQAGMTGYGMoxPR196 and ** denotes 183GApSQAGMoxTGYGMPR196.
Mentions: Our label-free quantitative phosphorylation analysis method was then used to investigate the phosphorylation level changes of GFP-tagged transgelin2 overexpressed in Jurkat T cells under PKC activation conditions. The most significant phosphorylation level change was observed with serine-185, in which the phosphorylation increased by more than two-fold. A manually validated MS/MS spectrum and selected ion chromatograms of the phosphopeptide containing serine-185 with or without PMA treatment are shown in Figure 2. Other phosphorylation sites showing significant phosphorylation level changes included serine-11 and threonine-180 (Additional file 4: Figure S3 and Additional file 5: Figure S4). Additionally, phosphopeptides containing methionine residue(s) were identified as discrete phosphopeptides, depending on the numbers and locations of oxidized methionine. For example, serine-185 co-localized with two methionine residues in the same tryptic digest peptide was identified in four different phosphopeptides. Two isobaric phosphopeptides, 183GAS*QAGMoxTGYGMPR196 and 183GAS*QAGMTGYGMoxPR196, whose only difference consisted of the location of the oxidized methionine, were clearly separated in the RPLC separation and unambiguously identified (Figure 3A and B). Finally, selected ion chromatograms of the phosphopeptides containing serine-185 with two oxidized methionines are shown in Figure 3C and D. The phosphorylation of transgelin2 under the PKA activation condition was also investigated by our quantitative phosphorylation analysis method. Although the number of phosphorylation sites and phosphopeptides identified were identical compared with those from the PKC activation, a moderate level of phosphorylation change (~36% increase) was observed only at serine-163 (Additional file 6: Figure S5). To increase the confidence level of our transgelin2 quantitative phosphorylation analysis results, the PKA, PKC and no activation condition experiments were independently performed three times. The peak areas of the individual phosphopeptides from the three independent experiments under the above two kinase activation conditions were calculated and plotted in terms of the peak area ratios compared with the no activation condition experiments (Figure 4 and Additional file 7: Figure S6). The preferential phosphorylation sites of transgelin2 under the PKA and PKC activation conditions appeared to vary. Serine-11, threonine-180 and serine-185 were responsive solely to PKC activation, whereas serine-163 is responsive to PKA activation.Figure 2

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