Profiling of human lymphocytes reveals a specific network of protein kinases modulated by endurance training status

To date, the effects of endurance exercise training on lymphocyte physiology at the kinome level are largely unknown. Therefore, the present study used a highly sensitive peptide-based kinase activity profiling approach to investigate if the basal activity of tyrosine (Tyr) and serine/threonine (Ser/Thr) kinases of human lymphocytes is affected by the aerobic endurance training status. Results revealed that the activity of various tyrosine kinases of the FGFR family and ZAP70 was increased, whereas the activity of multiple Ser/Thr kinases such as IKKα, CaMK4, PKAα, PKCα+δ (among others) was decreased in lymphocytes of endurance trained athletes (ET). Moreover, functional associations between several differentially regulated kinases in ET-derived lymphocytes were demonstrated by phylogenetic mapping and network analysis. Especially, Ser/Thr kinases of the AGC-kinase (protein kinase A, G, and C) family represent exercise-sensitive key components within the lymphocytes kinase network that may mediate the long-term effects of endurance training. Furthermore, KEGG (Kyoto Encyclopedia of Genes and Genomes) and Reactome pathway analysis indicate that Ras as well as intracellular signaling by second messengers were found to be enriched in the ET individuals. Overall, our data suggest that endurance exercise training improves the adaptive immune competence by modulating the activity of multiple protein kinases in human lymphocytes.


Supplementary information
. Raw data and heatmap representing the phosphorylation intensity of each peptide substrate spotted on the Tyr Pamchip®. The lymphocyte samples of the subjects are displayed as columns and the individual peptides are shown in the rows. The heatmap was created based on the log2 transformed raw data. Data was normalized via date centering. The peptides were ranked according to their correlation factor. The values of the phosphorylation intensities were translated into colors using a color scale. The scale for the phosphorylation intensity ranges from 1.2 (highest intensity, red color) to -1.2 (lowest intensity, blue color). AU = arbitrary units. Table S2. Raw data and heatmap representing the phosphorylation intensitiy of each peptide substrate spotted on the Ser/Thr Pamchip®. The lymphocyte samples of the subjects are displayed as columns and the individual peptides are shown in the rows. The heatmap was created based on the log2 transformed raw data. Data was normalized via date centering. The peptides were ranked according to their correlation factor. The values of the phosphorylation intensities were translated into colors using a color scale. The scale for the phosphorylation intensity ranges from 1 (highest intensity, red color) to -1 (lowest intensity, blue color). AU = arbitrary units.   Table S4. Kinase statistic of all serine/threonine kinases analyzed in this study. The constituents of the kinase statistic Specificity Score (Q sp ), Significance Score (Q sg ), Mean final score (Q mean ), Median Final Score (Q), Mean normalized kinase statistc (s mean ), Median normalized kinase statistic (s), Standard deviation of Kinase statistic (SD) and the mean peptide size are displayed as columns and the top ranked kinases marked by Unit-Prot IDs are presented in the rows. The final ranking of the kinases was based on Median Final Score (Q). Table S5. Raw data of each protein analyzed by Image J and the normalization procedure. and Ser/Thr kinases, which were associated with chronic endurance training (Q sp > 1.3). All input kinases (n = 15) match the highest-ranked term protein phosphorylation. Observed gene count: number of genes in the input data list with the term assigned; background gene count: total number of genes in the background proteome with the term assigne; FDR: false discovery rate     :0006468 protein phosphorylation  15,00  923,00  0,0000000  CAMK4,CHUK,CSNK2A1,FGFR1,FGFR2,FGFR3,FGFR4,PRKACA,PRKCA,PRKCD,PRKG1,PRKG2,PRKX,RPS6KB2,ZAP70  GO:0018193 peptidyl-amino acid modification  13,00  842,00  0,0000000  CAMK4,CHUK,CSNK2A1,FGFR1,FGFR2,FGFR3,FGFR4,

Tyrosine kinases FGFR
The specific role of FGFRs in human lymphocytes is poorly studied. FGFRs get activated by autophosphorylation after binding to their ligands FGFs, which were actively secreted under conditions related to strenuous endurance exercise such as hypoxia or ischemia 1,2 . Studies indicate that FGF-responsive T cells may play a role in the migration and proliferation of vascular-smooth muscle cells in vascular and inflammatory lesions 3 . The ligation of FGFR1 on T cells co-stimulates T cell receptor-triggered IL-2 production 4 .

ZAP70
ZAP-70 plays an essential role in lymphocyte activation by functioning in the initial step of T cell antigen receptor mediated signal transduction. Furthermore, ZAP-70 is involved in the regulation of adhesion, motility and cytokine expression of mature T cells and contributes to the activation and development of primary B cells. The phosphorylation of specific adapter proteins by ZAP70 leads to T cell proliferation, differentiation and the production of lymphokines https://www.uniprot.org/uniprot/P43403. T cells which lack ZAP-70 show defects in T cell activation and down-stream signaling events 5 .

Serine-/Threonine kinases PKG (PRKG)
PKGs are regulated by cGMP signaling. 6 . Protein targets for PKGs are involved in the regulation of cellular calcium homeostasis https://www.uniprot.org/uniprot/Q13976. Further, this kinase is positively associated with proteasome activities 10 . There is little information of the biological role of PKGs in human lymphocytes which is limited to its regulatory function in selected lymphocyte subpopulations. PKG1 affects the regulation of gene expression, is involved in the inhibition of T cell proliferation and controls the cytokine production of TH2 cells 7,8 . Moreover, the activation of PKG stimulates the down regulation of interleukin 2 signaling in T cell lines 9 .

IKKα (CHUK)
This kinase plays a crucial role in the NF-kappa-B signaling pathway which is stimulated by inflammatory cytokines, bacteria, viruses, DNA damage or other cellular stressors The IKK complex regulates genes encoding signaling molecules implicated in B cell survival as well as lymphoid organogenesis https://www.uniprot.org/uniprot/O15111. CAMK4 CAMK4 is a calcium/calmodulin-dependent multifunctional protein kinase that regulates gene expression by activating several transcription factors in T cells. In CD4 positive memory T-cells, CAMK4 is essential to link TCR signaling to the production of IL2, IFNγ and IL4 through the modulation of the transcription factors MEF2 and CREB https://www.uniprot.org/uniprot/Q16566. Besides, CAMK4 modulates several processes that contribute to the pathology of autoimmune diseases by regulating the production of IL-17 and IL-2 by T cells 11 . PKAα (PRKACA) The activity of PKAα depends on the cellular level of cAMP 6 . PKAα inhibits the early and late phase of the antigen-induced activation of T and B lymphocytes 12 . Furthermore, it contributes to the regulation of apoptosis of immature T lymphocytes 13 .

P70S6Kβ (RPS6SKB2)
Kinases of the P70S6K group affect important lymphocytic functions such as cell growth, differentiation, proliferation and function as a major regulator in glucose metabolism of T cells 14,15 .

PRKX
So far, the biological role of PRKX in lymphocytes is almost unknown due to a lack of studies 6,16 .

PKCα (PRKCA)
PKCα is activated by calcium and diacylglycerol (https://www.uniprot.org/uniprot/P17252). This kinase is involved in the activation of the IKK complex and NFκB in T lymphocytes as response to TCR activation 17 . Besides PKCα mediates TCR down regulation 18 .

CK2α1 (CSNK2A1)
CK2α1 phophorylates acidic proteins such as casein. This kinase regulates several cellular processes such as transcription, apoptosis and cell cycle progression https://www.uniprot.org/uniprot/P68400. Furthermore, it represents a crucial regulator of the balance of Th17 and Treg cell differentiation 19 .

PKCδ (PRKCD)
PKCδ is activated by diacylglycerol (https://www.uniprot.org/uniprot/Q05655). This isoform of protein kinase C is involved in the regulation of mitochondrial-dependent apoptosis via phosphorylation of histones at the apoptotic histone residues of T-Cells 20,21 . In addition, PKCδ plays a functional role in the regulation of apoptosis in resting B cells and suppresses the proliferation of B cells 22 .