scholarly journals An ultrasensitive fiveplex activity assay for cellular kinases

2019 ◽  
Author(s):  
Christian M. Smolko ◽  
Kevin A. Janes
Keyword(s):  
Protein Kinase ◽  
Protein Interactions ◽  
Posttranslational Modifications ◽  
Large Scale ◽  
Cell Types ◽  
Mitogen Activated Protein Kinase ◽  
Activity Measurement ◽  
Kinase Substrate ◽  
Iκb Kinase ◽  
Mitogen Activated Protein

ABSTRACTProtein kinases are enzymes whose abundance, protein-protein interactions, and posttranslational modifications together determine net signaling activity in cells. Large-scale data on cellular kinase activity are limited, because existing assays are cumbersome, poorly sensitive, low throughput, and restricted to measuring one kinase at a time. Here, we surmount the conventional hurdles of activity measurement with a multiplexing approach that leverages the selectivity of individual kinase-substrate pairs. We demonstrate proof of concept by designing an assay that jointly measures activity of five pleiotropic signaling kinases: Akt, IκB kinase (IKK), c-jun N-terminal kinase (JNK), mitogen-activated protein kinase (MAPK)-extracellular regulated kinase kinase (MEK), and MAPK-activated protein kinase-2 (MK2). The assay operates in a 96-well format and specifically measures endogenous kinase activation with coefficients of variation less than 20%. Multiplex tracking of kinase-substrate pairs reduces input requirements by 25-fold, with ~75 μg of cellular extract sufficient for fiveplex activity profiling. We applied the assay to monitor kinase signaling during coxsackievirus B3 infection of two different host-cell types and identified multiple differences in pathway dynamics and coordination that warrant future study. Because the Akt–IKK–JNK–MEK–MK2 pathways regulate many important cellular functions, the fiveplex assay should find applications in inflammation, environmental-stress, and cancer research.

scholarly journals An ultrasensitive fiveplex activity assay for cellular kinases

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Christian M. Smolko ◽  
Kevin A. Janes
Keyword(s):  
Protein Kinase ◽  
Protein Interactions ◽  
Posttranslational Modifications ◽  
Large Scale ◽  
Cell Types ◽  
Mitogen Activated Protein Kinase ◽  
Activity Measurement ◽  
Kinase Substrate ◽  
Iκb Kinase ◽  
Mitogen Activated Protein

AbstractProtein kinases are enzymes whose abundance, protein-protein interactions, and posttranslational modifications together determine net signaling activity in cells. Large-scale data on cellular kinase activity are limited, because existing assays are cumbersome, poorly sensitive, low throughput, and restricted to measuring one kinase at a time. Here, we surmount the conventional hurdles of activity measurement with a multiplexing approach that leverages the selectivity of individual kinase-substrate pairs. We demonstrate proof of concept by designing an assay that jointly measures activity of five pleiotropic signaling kinases: Akt, IκB kinase (IKK), c-jun N-terminal kinase (JNK), mitogen-activated protein kinase (MAPK)-extracellular regulated kinase kinase (MEK), and MAPK-activated protein kinase-2 (MK2). The assay operates in a 96-well format and specifically measures endogenous kinase activation with coefficients of variation less than 20%. Multiplex tracking of kinase-substrate pairs reduces input requirements by 25-fold, with ~75 µg of cellular extract sufficient for fiveplex activity profiling. We applied the assay to monitor kinase signaling during coxsackievirus B3 infection of two different host-cell types and identified multiple differences in pathway dynamics and coordination that warrant future study. Because the Akt–IKK–JNK–MEK–MK2 pathways regulate many important cellular functions, the fiveplex assay should find applications in inflammation, environmental-stress, and cancer research.

scholarly journals Selective expression of the scaffold protein JSAP1 in spermatogonia and spermatocytes

Reproduction ◽  
2006 ◽  
Vol 131 (4) ◽  
pp. 711-719 ◽  
Author(s):  
Munkhuu Bayarsaikhan ◽  
Akiko Shiratsuchi ◽  
Davaakhuu Gantulga ◽  
Yoshinobu Nakanishi ◽  
Katsuji Yoshioka
Keyword(s):  
Protein Kinase ◽  
Western Blotting ◽  
Cell Types ◽  
Mapk Signaling ◽  
Scaffold Protein ◽  
Mitogen Activated Protein Kinase ◽  
Interacting Protein ◽  
Early Postnatal Development ◽  
Mitogen Activated Protein ◽  
Signaling Modules

Scaffold proteins of mitogen-activated protein kinase (MAPK) intracellular signal transduction pathways mediate the efficient and specific activation of the relevant MAPK signaling modules. Previously, our group and others have identified c-Jun NH2-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1, also known as JNK-interacting protein 3) as a scaffold protein for JNK MAPK pathways. Although JSAP1 is expressed in the testis in adults, its expression during development has not been investigated. In addition, it is unknown which types of cells in the testis express the scaffold protein. Here, we examined the expression of JSAP1 in the testis of mice aged 14 days, 20 days, 6 weeks, and 12 weeks by immunohistochemistry and Western blotting. The specificity of the anti-JSAP1 antibody was evaluated from its reactivity to exogenously expressed JSAP1 and a structurally related protein, and by antigen-absorption experiments. The immunohistochemical analyses with the specific antibody showed that the JSAP1 protein was selectively expressed in the spermatogonia and spermatocytes, but not in other cell types, including spermatids and somatic cells, during development. However, not all spermatogonia and spermatocytes were immunopositive either, especially in the 12-week-old mouse testis. Furthermore, we found by Western blotting that the expression levels of JSAP1 protein vary during development; there is high expression until 6 weeks after birth, which approximately corresponds to the end of the first wave of spermatogenesis. Collectively, these results suggest that JSAP1 function may be important in spermatogenic cells during early postnatal development.

Regulation of MAP kinase by calcium-sensing receptor in bovine parathyroid and CaR-transfected HEK293 cells

2001 ◽  
Vol 280 (2) ◽  
pp. F291-F302 ◽  
Author(s):  
Olga Kifor ◽  
R. John MacLeod ◽  
Ruben Diaz ◽  
Mei Bai ◽  
Toru Yamaguchi ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Combined Treatment ◽  
Cell Types ◽  
Serine Phosphorylation ◽  
Mitogen Activated Protein Kinase ◽  
Hek293 Cells ◽  
Calcium Sensing ◽  
Cytosolic Phospholipase ◽  
Extracellular Signal ◽  
Mitogen Activated Protein

Regulation of the extracellular signal-regulated kinase 1 and 2 (ERK1/2) pathway by the extracellular calcium (Cao 2+)-sensing receptor (CaR) was investigated in bovine parathyroid and CaR-transfected human embryonic kidney (HEKCaR) cells. Elevating Cao 2+ or adding the selective CaR activator NPS R-467 elicited rapid, dose-dependent phosphorylation of ERK1/2. These phosphorylations were attenuated by pretreatment with pertussis toxin (PTX) or by treatment with the phosphotyrosine kinase (PTK) inhibitors genistein and herbimycin, the phosphatidylinositol-specific phospholipase C (PI-PLC) inhibitor U-73122, or the protein kinase C (PKC) inhibitor GF109203X and were enhanced by the PKC activator phorbol 12-myristate 13-acetate. Combined treatment with PTX and inhibitors of both PKC and PTK nearly abolished high Cao 2+-evoked ERK1/2 activation in HEKCaR cells, demonstrating CaR-mediated coupling via both Gq and Gi. High Cao 2+ increased serine phosphorylation of the 85-kDa cytosolic phospholipase A2(cPLA2) in both parathyroid and HEKCaR cells. The selective mitogen-activated protein kinase (MAPK) inhibitor PD98059 abolished high-Cao 2+-induced ERK1/2 activation and reduced cPLA2 phosphorylation in both cell types, documenting MAPK's role in cPLA2 activation. Thus our data suggest that the CaR activates MAPK through PKC, presumably through Gq/11-mediated activation of PI-PLC, as well as through Gi- and PTK-dependent pathway(s) in bovine parathyroid and HEKCaR cells and indicate the importance of MAPK in cPLA2 activation.

Non-Genomic Effects of Estradiol on Human Platelets: Regulation of Mitogen Activated Protein Kinase Activation and Enhanced Spreading and Aggregation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2620-2620
Author(s):  
Kadekuzhi V. Vijayan ◽  
Yan Liu ◽  
Tong-Tong Li ◽  
Paul F. Bray
Keyword(s):  
Bone Marrow ◽  
Platelet Aggregation ◽  
Protein Kinase ◽  
Randomized Clinical Trials ◽  
Hormone Replacement ◽  
Cell Types ◽  
Human Platelets ◽  
Mitogen Activated Protein Kinase ◽  
Induce Platelet Aggregation ◽  
Mitogen Activated Protein

Abstract 17β-estradiol or estrogen (E2) is a sex hormone that modulates platelet function and is widely used in hormone replacement therapy (HRT). We and others have previously demonstrated that human megakaryocytes and platelets posses estrogen receptors ERα and ERβ. HRT treatment augmented the bone marrow megakaryocytes without increasing other bone marrow cells, suggesting that E2 can modulate proliferation of megakaryocytes. Since mitogen activated protein kinases (MAPKs) are critical for 1) cell proliferation, 2) megakarytocyte differentiation and proplatelet formation and estrogen activates MAPK in other cell types, we hypothesized that estrogen regulates the activation of MAPK in human platelets. Signaling was studied using washed platelets from male and female subjects in response to varying concentrations of estrogen. Compared to the ethanol (vehicle) treated platelets, 1 nM E2 treated platelets for 60 seconds resulted in an enhanced activation of extracellular signal-regulated kinase 2 (ERK 2) and P38 but not Jun N-kinase (JNK). These results suggest that E2 can cause a non-genomic signaling in human platelets. The MEK inhibitors PD98059 and U0216 blocked the E2 effect, suggesting that the activation of ERK 2 was mediated through the upstream mitogen activated protein kinase kinase (MAPKK). Because E2 can modulate actin reorganization in other cell types and cell spreading is promoted by ERK 2 activation, we examined the effect of E2 on platelet spreading - a process not dependent on agonist stimulation. Compared to ethanol treated platelets, platelets preincubated with 100 nM E2 for 60 seconds and stained with rhodamine phallodine exhibited a ~60 % greater spreading at 5 and 15 minutes. This observation suggests that that E2 can cause rapid actin cytoskeletal reorganization in platelets. Since inhibition of ERK 2 activation blocks aggregation to low doses of thrombin and collagen, we examined a role for E2 in platelet aggregation. E2 alone did not induce platelet aggregation. However, E2 potentiated aggregation with low but not high doses of thrombin and collagen related peptide (CRP) (P=0.05 for 0.02 μg/ml thrombin and P<0.001 for 0.2 μg/ml CRP). Our data demonstrates that E2 can activate MAPK through a non-genomic mechanism and this activation correlates with greater platelet functions like spreading and aggregation. Our findings support a mechanism whereby a consistent non-genomic enhancement of platelet signaling and reactivity by E2 may underlie the increased cardiovascular events observed in recent randomized clinical trials with HRT.

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