Table2_Tyrosine Phosphorylation Profiling Revealed the Signaling Network Characteristics of CAMKK2 in Gastric Adenocarcinoma.PDF

Calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) is a serine/threonine protein kinase which functions via the calcium-triggered signaling cascade with CAMK1, CAMK4, and AMPKα as the immediate downstream substrates. CAMKK2 is reported to be overexpressed in gastric cancer; however, its signaling mechanism is poorly understood. We carried out label-free quantitative tyrosine phosphoproteomics to investigate tyrosine-mediated molecular signaling associated with CAMKK2 in gastric cancer cells. Using a high-resolution Orbitrap Fusion Tribrid Fourier-transform mass spectrometer, we identified 350 phosphotyrosine sites mapping to 157 proteins. We observed significant alterations in 81 phosphopeptides corresponding to 63 proteins upon inhibition of CAMKK2, among which 16 peptides were hyperphosphorylated corresponding to 13 proteins and 65 peptides were hypophosphorylated corresponding to 51 proteins. We report here that the inhibition of CAMKK2 leads to changes in the phosphorylation of several tyrosine kinases such as PKP2, PTK2, EPHA1, EPHA2, PRKCD, MAPK12, among others. Pathway analyses revealed that proteins are differentially phosphorylated in response to CAMKK2 inhibition involved in focal adhesions, actin cytoskeleton, axon guidance, and signaling by VEGF. The western blot analysis upon inhibition and/or silencing of CAMKK2 revealed a decrease in phosphorylation of PTK2 at Y925, c-JUN at S73, and STAT3 at Y705, which was in concordance with the mass spectrometry data. The study indicates that inhibition of CAMKK2 has an anti-oncogenic effect in gastric cells regulating phosphorylation of STAT3 through PTK2/c-JUN in gastric cancer.</p

Image5_Tyrosine Phosphorylation Profiling Revealed the Signaling Network Characteristics of CAMKK2 in Gastric Adenocarcinoma.TIF

Calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) is a serine/threonine protein kinase which functions via the calcium-triggered signaling cascade with CAMK1, CAMK4, and AMPKα as the immediate downstream substrates. CAMKK2 is reported to be overexpressed in gastric cancer; however, its signaling mechanism is poorly understood. We carried out label-free quantitative tyrosine phosphoproteomics to investigate tyrosine-mediated molecular signaling associated with CAMKK2 in gastric cancer cells. Using a high-resolution Orbitrap Fusion Tribrid Fourier-transform mass spectrometer, we identified 350 phosphotyrosine sites mapping to 157 proteins. We observed significant alterations in 81 phosphopeptides corresponding to 63 proteins upon inhibition of CAMKK2, among which 16 peptides were hyperphosphorylated corresponding to 13 proteins and 65 peptides were hypophosphorylated corresponding to 51 proteins. We report here that the inhibition of CAMKK2 leads to changes in the phosphorylation of several tyrosine kinases such as PKP2, PTK2, EPHA1, EPHA2, PRKCD, MAPK12, among others. Pathway analyses revealed that proteins are differentially phosphorylated in response to CAMKK2 inhibition involved in focal adhesions, actin cytoskeleton, axon guidance, and signaling by VEGF. The western blot analysis upon inhibition and/or silencing of CAMKK2 revealed a decrease in phosphorylation of PTK2 at Y925, c-JUN at S73, and STAT3 at Y705, which was in concordance with the mass spectrometry data. The study indicates that inhibition of CAMKK2 has an anti-oncogenic effect in gastric cells regulating phosphorylation of STAT3 through PTK2/c-JUN in gastric cancer.</p

Image2_Tyrosine Phosphorylation Profiling Revealed the Signaling Network Characteristics of CAMKK2 in Gastric Adenocarcinoma.TIF

Calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) is a serine/threonine protein kinase which functions via the calcium-triggered signaling cascade with CAMK1, CAMK4, and AMPKα as the immediate downstream substrates. CAMKK2 is reported to be overexpressed in gastric cancer; however, its signaling mechanism is poorly understood. We carried out label-free quantitative tyrosine phosphoproteomics to investigate tyrosine-mediated molecular signaling associated with CAMKK2 in gastric cancer cells. Using a high-resolution Orbitrap Fusion Tribrid Fourier-transform mass spectrometer, we identified 350 phosphotyrosine sites mapping to 157 proteins. We observed significant alterations in 81 phosphopeptides corresponding to 63 proteins upon inhibition of CAMKK2, among which 16 peptides were hyperphosphorylated corresponding to 13 proteins and 65 peptides were hypophosphorylated corresponding to 51 proteins. We report here that the inhibition of CAMKK2 leads to changes in the phosphorylation of several tyrosine kinases such as PKP2, PTK2, EPHA1, EPHA2, PRKCD, MAPK12, among others. Pathway analyses revealed that proteins are differentially phosphorylated in response to CAMKK2 inhibition involved in focal adhesions, actin cytoskeleton, axon guidance, and signaling by VEGF. The western blot analysis upon inhibition and/or silencing of CAMKK2 revealed a decrease in phosphorylation of PTK2 at Y925, c-JUN at S73, and STAT3 at Y705, which was in concordance with the mass spectrometry data. The study indicates that inhibition of CAMKK2 has an anti-oncogenic effect in gastric cells regulating phosphorylation of STAT3 through PTK2/c-JUN in gastric cancer.</p

Image4_Tyrosine Phosphorylation Profiling Revealed the Signaling Network Characteristics of CAMKK2 in Gastric Adenocarcinoma.TIF

Calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) is a serine/threonine protein kinase which functions via the calcium-triggered signaling cascade with CAMK1, CAMK4, and AMPKα as the immediate downstream substrates. CAMKK2 is reported to be overexpressed in gastric cancer; however, its signaling mechanism is poorly understood. We carried out label-free quantitative tyrosine phosphoproteomics to investigate tyrosine-mediated molecular signaling associated with CAMKK2 in gastric cancer cells. Using a high-resolution Orbitrap Fusion Tribrid Fourier-transform mass spectrometer, we identified 350 phosphotyrosine sites mapping to 157 proteins. We observed significant alterations in 81 phosphopeptides corresponding to 63 proteins upon inhibition of CAMKK2, among which 16 peptides were hyperphosphorylated corresponding to 13 proteins and 65 peptides were hypophosphorylated corresponding to 51 proteins. We report here that the inhibition of CAMKK2 leads to changes in the phosphorylation of several tyrosine kinases such as PKP2, PTK2, EPHA1, EPHA2, PRKCD, MAPK12, among others. Pathway analyses revealed that proteins are differentially phosphorylated in response to CAMKK2 inhibition involved in focal adhesions, actin cytoskeleton, axon guidance, and signaling by VEGF. The western blot analysis upon inhibition and/or silencing of CAMKK2 revealed a decrease in phosphorylation of PTK2 at Y925, c-JUN at S73, and STAT3 at Y705, which was in concordance with the mass spectrometry data. The study indicates that inhibition of CAMKK2 has an anti-oncogenic effect in gastric cells regulating phosphorylation of STAT3 through PTK2/c-JUN in gastric cancer.</p

Table1_Tyrosine Phosphorylation Profiling Revealed the Signaling Network Characteristics of CAMKK2 in Gastric Adenocarcinoma.PDF

Calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) is a serine/threonine protein kinase which functions via the calcium-triggered signaling cascade with CAMK1, CAMK4, and AMPKα as the immediate downstream substrates. CAMKK2 is reported to be overexpressed in gastric cancer; however, its signaling mechanism is poorly understood. We carried out label-free quantitative tyrosine phosphoproteomics to investigate tyrosine-mediated molecular signaling associated with CAMKK2 in gastric cancer cells. Using a high-resolution Orbitrap Fusion Tribrid Fourier-transform mass spectrometer, we identified 350 phosphotyrosine sites mapping to 157 proteins. We observed significant alterations in 81 phosphopeptides corresponding to 63 proteins upon inhibition of CAMKK2, among which 16 peptides were hyperphosphorylated corresponding to 13 proteins and 65 peptides were hypophosphorylated corresponding to 51 proteins. We report here that the inhibition of CAMKK2 leads to changes in the phosphorylation of several tyrosine kinases such as PKP2, PTK2, EPHA1, EPHA2, PRKCD, MAPK12, among others. Pathway analyses revealed that proteins are differentially phosphorylated in response to CAMKK2 inhibition involved in focal adhesions, actin cytoskeleton, axon guidance, and signaling by VEGF. The western blot analysis upon inhibition and/or silencing of CAMKK2 revealed a decrease in phosphorylation of PTK2 at Y925, c-JUN at S73, and STAT3 at Y705, which was in concordance with the mass spectrometry data. The study indicates that inhibition of CAMKK2 has an anti-oncogenic effect in gastric cells regulating phosphorylation of STAT3 through PTK2/c-JUN in gastric cancer.</p

PfPK2 depletion impairs the entry of merozoites in host erythrocytes.

A. Schizonts isolated from PfPK2-loxP parasites cultured in the presence or absence (DMSO) of RAP for one cycle and post-egress merozoites were used for live microscopy (S1–S4 Video). Selected still images from videos are used to illustrate various stages of invasion. DMSO-treated PfPK2-loxP merozoites successfully deform the erythrocyte membrane (red arrow), invade, and trigger echinocytosis (white arrow) followed by recovery of the infected erythrocyte (a, video S1). Prolonged attachment upon RAP-treatment and unsuccessful invasion was observed (b, video S2). Several RAP-treated parasites that exhibited prolonged attachment (red arrow) were also able to deform the erythrocyte membrane (c, S3 Video) and stimulate echinocytosis (EC) which was for much longer duration (d, white arrow, S4 Video). B. % invasion per attachment was determined and data represent results from four independent pooled experiments described in panel A (mean ± SE, n  =  4, *, P C. Various outcomes during erythrocyte invasion indicated in Panel A were quantitated for merozoites that formed an initial attachment using data from four different experiments for each set of conditions [****, P < 0.0001, chi-square test].</p

Image6_Tyrosine Phosphorylation Profiling Revealed the Signaling Network Characteristics of CAMKK2 in Gastric Adenocarcinoma.TIF

Calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) is a serine/threonine protein kinase which functions via the calcium-triggered signaling cascade with CAMK1, CAMK4, and AMPKα as the immediate downstream substrates. CAMKK2 is reported to be overexpressed in gastric cancer; however, its signaling mechanism is poorly understood. We carried out label-free quantitative tyrosine phosphoproteomics to investigate tyrosine-mediated molecular signaling associated with CAMKK2 in gastric cancer cells. Using a high-resolution Orbitrap Fusion Tribrid Fourier-transform mass spectrometer, we identified 350 phosphotyrosine sites mapping to 157 proteins. We observed significant alterations in 81 phosphopeptides corresponding to 63 proteins upon inhibition of CAMKK2, among which 16 peptides were hyperphosphorylated corresponding to 13 proteins and 65 peptides were hypophosphorylated corresponding to 51 proteins. We report here that the inhibition of CAMKK2 leads to changes in the phosphorylation of several tyrosine kinases such as PKP2, PTK2, EPHA1, EPHA2, PRKCD, MAPK12, among others. Pathway analyses revealed that proteins are differentially phosphorylated in response to CAMKK2 inhibition involved in focal adhesions, actin cytoskeleton, axon guidance, and signaling by VEGF. The western blot analysis upon inhibition and/or silencing of CAMKK2 revealed a decrease in phosphorylation of PTK2 at Y925, c-JUN at S73, and STAT3 at Y705, which was in concordance with the mass spectrometry data. The study indicates that inhibition of CAMKK2 has an anti-oncogenic effect in gastric cells regulating phosphorylation of STAT3 through PTK2/c-JUN in gastric cancer.</p

Image1_Tyrosine Phosphorylation Profiling Revealed the Signaling Network Characteristics of CAMKK2 in Gastric Adenocarcinoma.TIF

Calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) is a serine/threonine protein kinase which functions via the calcium-triggered signaling cascade with CAMK1, CAMK4, and AMPKα as the immediate downstream substrates. CAMKK2 is reported to be overexpressed in gastric cancer; however, its signaling mechanism is poorly understood. We carried out label-free quantitative tyrosine phosphoproteomics to investigate tyrosine-mediated molecular signaling associated with CAMKK2 in gastric cancer cells. Using a high-resolution Orbitrap Fusion Tribrid Fourier-transform mass spectrometer, we identified 350 phosphotyrosine sites mapping to 157 proteins. We observed significant alterations in 81 phosphopeptides corresponding to 63 proteins upon inhibition of CAMKK2, among which 16 peptides were hyperphosphorylated corresponding to 13 proteins and 65 peptides were hypophosphorylated corresponding to 51 proteins. We report here that the inhibition of CAMKK2 leads to changes in the phosphorylation of several tyrosine kinases such as PKP2, PTK2, EPHA1, EPHA2, PRKCD, MAPK12, among others. Pathway analyses revealed that proteins are differentially phosphorylated in response to CAMKK2 inhibition involved in focal adhesions, actin cytoskeleton, axon guidance, and signaling by VEGF. The western blot analysis upon inhibition and/or silencing of CAMKK2 revealed a decrease in phosphorylation of PTK2 at Y925, c-JUN at S73, and STAT3 at Y705, which was in concordance with the mass spectrometry data. The study indicates that inhibition of CAMKK2 has an anti-oncogenic effect in gastric cells regulating phosphorylation of STAT3 through PTK2/c-JUN in gastric cancer.</p

Time-lapse video microscopy of RAP- treated PfPK2-loxP parasites (related to Fig 4Ab).

Time-lapse video microscopy of RAP- treated PfPK2-loxP parasites (related to Fig 4Ab).</p

PCR Primers used in this study.

Signalling pathways in malaria parasite remain poorly defined and major reason for this is the lack of understanding of the function of majority of parasite protein kinases and phosphatases in parasite signalling and its biology. In the present study, we have elucidated the function of Protein Kinase 2 (PfPK2), which is known to be indispensable for the survival of human malaria parasite Plasmodium falciparum. We demonstrate that it is involved in the invasion of host erythrocytes, which is critical for establishing infection. In addition, PfPK2 may also be involved in the maturation of the parasite post-invasion. PfPK2 regulates the release of microneme proteins like Apical Membrane Antigen 1 (AMA1), which facilitates the formation of Tight Junction between the merozoite and host erythrocyte- a key step in the process of invasion. Comparative phosphoproteomics studies revealed that PfPK2 may be involved in regulation of several key proteins involved in invasion and signalling. Furthermore, PfPK2 regulates the generation of cGMP and the release of calcium in the parasite, which are key second messengers for the process of invasion. These and other studies have shed light on a novel signalling pathway in which PfPK2 acts as an upstream regulator of important cGMP-calcium signalling, which plays an important role in parasite invasion.</div