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Pharmacology & Toxicology Poster Session






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Tyrosine Kinase-Mediated Serine Phosphorylation and Enhancement of Adenylyl Cyclase Activity


Contact Person: Christopher M. Tan (ctan@julian.uwo.ca)


Results

Sham or FAC6-transfected HEK 293 cells were permeabilized and subsequently assayed for GTP (10 Ámol/L)-, Isoproterenol+GTP (100 Ámol/L + 10 Ámol/L, Iso+GTP)- and forskolin (10 Ámol/L)-stimulated adenylyl cyclase activities. Data are compared to their corresponding controls, where each control represents stimulated adenylyl cyclase activity in sham HEK 293 cells. Data is expressed as pmol/min/mg protein. Data represents the arithmetic mean " SEM for six experiments performed with each stimulator. *p < 0.05 vs. corresponding control stimulated activity (Figure 1A). HEK 293 cells were transfected with 10 Ág pRc/CMV-FAC6 and maintained for 72 hours. FAC6-transfected HEK 293 cells were lysed, FAC6 immunoprecipitated and resolved via SDS-PAGE. Proteins were transferred to membranes and immunoblotted with either anti-Flag M5 (upper panel) or anti AC COMM (lower panel). Lane 1: 100 ng recombinant FAC6 protein standard; Lanes 2,3,4: Crude lysate (C), Immunoprecipitated FAC6 (I), post-immunoprecipitated Supernatant (S), respectively (Figure 1B).

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Figure 1: The effect of FAC6 transient transfection in HEK 293 cells. A) Functional Analysis. B) Immunoblot analysis.

Permeabilized FAC6-transfected HEK 293 cells were treated with varying concentrations of vanadate and adenylyl cyclase activity was assessed. Each data point represents the geometric mean " SEM of five experiments. (Figure 2A). Vanadate-mediated alterations in GTP (10 Ámol/L)-, Isoproterenol+GTP (100 Ámol/L + 10 Ámol/L, Iso+GTP)- and forskolin (10 Ámol/L)-stimulated activities are compared to their corresponding controls, where each control represents stimulated adenylyl cyclase activity in the absence of vanadate (Figure 2B). The effect of vanadate on adenylyl cyclase activity was tyrosine kinase dependent. FAC6-transfected HEK 293 cells were permeabilized and treated with either the active tyrosine kinase inhibitor Tyrphostin B46 ( 40 Ámol/L) or its structurally similar but chemically inactive inhibitor analog Tyrphostin A1 (100 Ámol/L) for 15 minutes. Forskolin-stimulated adenylyl cyclase activity was assessed in the presence or absence of vanadate (100 Ámol/L). Data is expressed as pmol/min/mg protein (Figure 2C). Data represents the arithmetic mean " SEM for four (Figure 2B) and six (Figure 2C) experiments performed with each stimulator. *p < 0.05 vs. corresponding control stimulated activity.

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Figure 2: Effect of vanadate on adenylyl cyclase activity in FAC6-transfected HEK 293 cells: Functional Analysis. A) Dose dependent enhancement of adenylyl cyclase activity. B) Effect of vanadate on stimulated adenylyl cyclase activity. C) Effect of tyrosine kinase inhibition on the vanadate-mediated enhancement of adenylyl cyclase activity.

The effect of vanadate on [32P]phosphate incorporation into adenylyl cyclase was assessed. FAC6-transfected HEK 293 cells were metabolically labeled with [32P]orthophosphate and treated in the absence or presence of vanadate (300 Ámol/L). FAC6 immunoprecipitated from control (-) or vanadate-treated (+) FAC6-transfected HEK 293 cells was resolved via SDS-PAGE (Figure 3A, upper panel). Resolved anti-Flag immunoprecipitates probed using anti-Flag antibody demonstrated that FAC6 protein content was similar in each lane (Figure 3A, lower panel). The vanadate-mediated phosphorylation was tyrosine kinase dependent (Figure 3B). In the presence of [32P]orthophosphate, FAC6-transfected HEK 293 cells were left untreated or pretreated with or without Tyrphostin B46 (100 Ámol/L) or Tyrphostin A1 (100 Ámol/L) in the presence or absence of vanadate (300 Ámol/L) and handled as above. Data is expressed as % of control, where control represents [32P]phosphate incorporation in the absence of vanadate and inhibitors in arbitrary densitometric units. Data represents the geometric mean " SEM from twelve experiments performed under identical conditions. *p < 0.05 vs. corresponding control [32P]phosphate incorporation.

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Figure 3: Effect of vanadate on adenylyl cyclase activity in FAC6-transfected HEK 293 cells. A) Phosphorylation/Immunoblot Analysis. B) Densitometric Analysis.

To examine the identity of the phosphoacceptor amino acid sites, FAC6 immunoprecipitates were excised from the gel (inset), FAC6 precipitated, hydrolyzed and subjected to 2-dimensional electrophoresis on TLC plates (left margin). The relative migration pattern of cold phosphoamino acid standards is depicted (right margin). Vanadate mediated enhanced serine phosphorylation of FAC6 (Panel B vs. Panel A, Figure 4). In contrast, vanadate did not alter the phosphothreonine nor phosphotyrosine content. The vanadate-mediated serine phosphorylation was tyrosine kinase dependent. The effect of vanadate to enhance phosphoserine content was abrogated by tyrosine kinase inhibition (Panel D).

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Figure 4: Effect of vanadate on adenylyl cyclase activity in FAC6-transfected HEK 293 cells: Phosphoamino Acid Analysis. A representative series of autoradiographs depicting the amino acid hydrolysis of FAC6 under control (A) and vanadate-treated (B) conditions in the absence (C) and presence (D) of Tyrphostin B46.

To examine potential serine/threonine kinases which may be involved in the vanadate-mediated serine phosphorylation of Flag epitope-tagged adenylyl cyclase, several serine/threonine kinase inhibitors were utilized. Staurosporine (1 Ámol/L, a kinase inhibitor which displays a broad serine/threonine kinase inhibition) significantly attenuated the vanadate-mediated enhancement of adenylyl cyclase activity (Table).

INHIBITOR      CONCENTRATION        VANADATE-MEDIATED ENHANCEMENT 
               (Ámol/L)             OF ADENYLYL CYCLASE ACTIVITY
                                   (% of Control)
----------------------------------------------------------------

Control                            100.0
Wortmannin        0.1               99.8 ▒ 7.8
PD98059+        100.0,100.0         95.1 ▒ 4.7
  Olomoucine
H89               0.6              100.5 ▒ 2.9
H8              100.0               95.3 ▒ 2.8
Staurosporine     1.0               71.5 ▒ 7.5 *
Values are mean ▒ SEM for forskolin-stimulated adenylyl cyclase activity from five experiments performed under identical conditions. Data is expressed as % of control, where control represents vanadate-mediated enhancement of adenylyl cyclase activity in the absence (Control) of inhibitors. Comparison between Control and individual inhibitor groups was performed by repeated measures ANOVA followed by Dunnettĺs multiple comparisons test. *p < 0.05 vs. corresponding control. To determine if the vanadate-mediated serine phosphorylation was required for the vanadate-mediated enhancement of adenylyl cyclase activity, candidate serine residues located within the catalytic C1b loop were selectively mutated to alanine residues via PCR-based site-directed mutagenesis using FAC6 as the wild type template (Figure 5). Initial studies demonstrated that each FAC6 mutant was expressed to comparable levels with respect to FAC6 protein (as assessed via immunoblotting) and that forskolin-stimulated adenylyl cyclase activity of each mutant was identical to that of FAC6 (data not shown).

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Figure 5: Site directed mutagenesis: Candidate serine residues.

Permeabilized FAC6-, S588A-, S6038A-, AND S7454A- transfected HEK 293 cells were treated in the absence or presence of vanadate (300 Ámol/L) and forskolin-stimulated adenylyl cyclase activity was assessed. Data is expressed as % inhibition of vanadate-mediated enhancement of adenylyl cyclase activity in the heterologous expression system (Figure 6A), or corrected for transfection efficiency (Figure 6A). Data represents the geometric mean " SEM for seven experiments performed under identical conditions. *p < 0.05 vs. corresponding control stimulated activity.

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Figure 6: Effect of vanadate on adenylyl cyclase activity in FAC6- 588A-, S6038A-, or S7454A-transfected HEK 293 cells: Functional Studies.

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Tan, CM; Feldman, RD; (1998). Tyrosine Kinase-Mediated Serine Phosphorylation and Enhancement of Adenylyl Cyclase Activity. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Available at URL http://www.mcmaster.ca/inabis98/pharmtox/tan0766/index.html
© 1998 Author(s) Hold Copyright