Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References

Discussion
Board

Sham or FAC6-transfected HEK 293 cells were permeabilized and subsequently assayed for GTP (10 µmol/L)-, Isoproterenol+GTP (100 µmol/L + 10 µmol/L, Iso+GTP)- and forskolin (10 µmol/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 µg 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).

click to enlarge

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 µmol/L)-, Isoproterenol+GTP (100 µmol/L + 10 µmol/L, Iso+GTP)- and forskolin (10 µmol/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 µmol/L) or its structurally similar but chemically inactive inhibitor analog Tyrphostin A1 (100 µmol/L) for 15 minutes. Forskolin-stimulated adenylyl cyclase activity was assessed in the presence or absence of vanadate (100 µmol/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.

click to enlarge

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 µmol/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 µmol/L) or Tyrphostin A1 (100 µmol/L) in the presence or absence of vanadate (300 µmol/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.

click to enlarge

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).

click to enlarge

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 µmol/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 
               (µmol/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 *

click to enlarge

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 µmol/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.

click to enlarge

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

Back to the top.