SYNAPSE 5:241-246 (1990)
Cyclic AMP-Dependent Protein PhosphorylaGon in the Rat Anterior Pituitary SCOTT T. CAIN, JAMES CLIFF PRYOR, AND CHARLES B. NEMEROFF Departments of Psychiatry (S.T.C., J.C.P., C.B.N.) and Pharmacology (C.B.N.), Duke University Medical Center, Durham, North Carolina 27710
KEY WORDS
Cyclic AMP, Protein phosphorylation, Anterior pituitary, Protein kinase
ABSTRACT The activation of cyclic adenosine 3'5'-monophosphate (CAMP)-dependent protein kinases has been implicated as an integral mechanism in stimulus-secretion coupling in the anterior pituitary. Therefore, we have investigated phosphorylation of endogenous protein substrates both in the presence and absence of CAMPin cell-free extracts of the rodent anterior pituitary. Specific phosphoprotein substrates in the rat anterior pituitary, which are phosphorylated by a CAMP-dependent protein kinase in vitro, were identified. Cyclic AMP potentiated the phosphorylation of proteins with apparent molecular weights of 85,000, 77,000, 63,000, 53,000, 39,000, and 33,000 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins with apparent molecular weights of 124,000, 93,000, 48,000, and 43,000 were phosphorylated only in the presence of CAMPand not in the basal condition. The results highlight endogenous protein substrates that may potentially be involved in CAMPdependent stimulus-secretion coupling in the anterior pituitary. INTRODUCTION The receptor-coupled stimulation of CAMPformation and the concomitant activation of CAMP-dependentprotein kinases is believed to be an important exocytotic regulatory mechanism in a number of biological systems (for review see Harper, 1988). In particular, considerable evidence supports the notion that activation of CAMP-dependent protein kinases is a crucial intermediary step in secretagogue-induced release of adenohypophyseal hormones from the anterior pituitary. For example, corticotropin-releasing factor (CRF), the primary physiological regulator of adrenocorticotropin release, activates adenylate cyclase and thus stimulates CAMPformation in both rodent (Aguilera et al., 1983, 1986; Labrie et al., 1982; Sobel, 1985) and primate (Millan et al., 1987) anterior pituitary cells. The doseresponse of CRF-stimulated CAMP formation and CAMP-dependent protein kinase activity closely parallels CRF-stimulated ACTH release (Litvin et al., 1984). Moreover, other hypothalamic hypophysiotrophic hormones such as vasoactive intestinal peptide (Gourdji et al., 19851,growth hormone-releasing factor (Schettini et al., 19841, and luteinizing hormone-releasing hormone (Labrie et al., 1979) are believed to produce their effects on anterior pituitary tro ic hormone release by CAMP-mediated mechanisms. yclic AMP-responsive phosphoprotein substrates have previously been identified in a clonal GH pituitary cell line (Drust et al., 19821, bovine anterior pituitary land (LeMayet al., 19741,and AtT-20/D16-16 tumor ce 1s (Pasmantier et al., 1986; Rougon et al., 1989). However, in spite of this rich literature, little is known of the identity of the CAMP-dependent phos-
e
7
0 1990 WILEY-LISS,INC.
phoprotein substrates in the rodent anterior pituitary, which, in our laboratory and others, is commonly used as a model system to study the hypothalamic-pituitary axis. We have, therefore, examined CAMP-dependent protein phosphorylation in cell-free extracts of the rat anterior pituitary. Although a previous attempt to identify CAMP-dependent phosphoprotein substrates in vitro in the rat anterior pituitar was not successful (Brattin and Portanova, 1981))we ave now identified a number of specific rat anterior pituitary proteins that are responsive to CAMP. MATERIALS AND METHODS Materials Rats were obtained from the Charles River colony in (specific activity 4-5 X Raleigh, N.C. [Y~~PI-ATP lo3 Ci/mM) was obtained from ICN. Electrophoresis equipment and reagants were from Bio-Rad, CAMPwas obtained from Sigma Chemicals, and all other chemicals were reagant grade. Tissue preparation and in vitro phosphorylation Adult male Sprague-Dawley rats were decapitated and the brains rapidly removed to expose the pituitary glands. The anterior pituitary was then separated from the neurointermediate and posterior lobes and homogenized in 20 volumes of 150 mM HEPES, 24 mM MgC12,
K
Received April 27,1989; accepted October 18,1989 Address reprint requests to S. T. Cain, Department of Psychiatry, Box 3859, Duke University Medical Center, Durham, North Carolina 27710.
242
S.T. CAIN ET AL.
and 0.5 mM EGTA, pH 7.1. In vitro phosphorylation was performed using a minor modification of a standard protocol designed to analyze cyclic nucleotide-dependent phosphorylation of endogenous protein substrates (see, e.g., Rudolph and Krueger, 1979). Aliquots of the tissue homogenate were preincubated a t 30°C. After 40 seconds, CAMPor vehicle was added. Twenty seconds later, the phosphorylation reaction was initiated by the addition of ATP (containing [Y~~PI-ATP, approximately 7 X lo6 cpdsample) (final concentrations: 50 mM HEPES, 8 mM MgCl,, 0.17 mM EGTA, 10 pM ATP with or without 1-100 pM CAMP).After 40 seconds of phosphorylation, an aliquot of the sample was removed for trichloroacetic acid (TCA) precipitation on filter papers and liquid scintillation counting. Phosphorylation was allowed to proceed for 60 seconds before being quenched with sodium dodecyl sulfate (SDS) stop solution (final concentration: 3% SDS, 2% P-mercaptoethanol, 1.7%glycerol, 0.01 M Tris pH 8.9). Electrophoresis, autoradiography and densitometric analysis Phosphorylated samples were boiled for 3 minutes at 80°C prior to electrophoresis. Equal amounts of sample proteins were separated on SDS-polyacrylamide gels according to the method of Krueger et al. (1977). The resolving gel consisted of 10% or 7.5% acrylamide, 0.27% bis-acrylamide, 0.375 M Tris pH 8.9, 0.1% N, N, N’, N’ tetramethylethylenediamine (TEMED), and 0.1% SDS. The stacking gel consisted of 5% acrylamide, 0.13% bis-acrylamide, 0.125 M Tris pH 6.8, 0.1% TEMED, and 0.1% SDS. Polymerization of both stacking and resolving gels was initiated by the addition of 0.15% ammonium persulfate. Electrophoresis buffer, both upper and lower chambers, was 0.05 M Tris pH 8.3, 0.375 Mglycine, and 0.1%SDS. Electrophoresis was run at 25 &gel for approximately 1 hour and then at 35 &gel until the bromphenol blue tracking dye was at the bottom of the gel. The gels were fixed and stained in acetic acid-methanol solution containing coomassie blue, dried under vacuum, and autoradiograms prepared by exposing the dried gels to Kodak NS-5T x-ray film for 14 days. Phosphate incorporation into individual proteins was quantitated by microdensitometric analysis of the autoradiograms. Peak height above background was measured for each phosphoprotein. Peak heights for all phosphoproteins within a sample were cumulated to quantitate the “total” hosphorylation in that sample. Molecular weights of p osphorylated proteins were determined by comparison t,o the migration of proteins of known molecular weight. Statistical analysis Vehicle and CAMP-dosle response groups were subjected to a one-way analysis of variance. If the probability of the F score was less than 0.05, dose-response effects were compared to the control level of phosphorylation using Dunnett’s test. Differences were considered statistically significant if p < .05. RESULTS Rat anterior pituitary extracts were phosphorylated with [y3’P]-ATP. As can be seen in the autoradiograph provided in Figure 1 (lanes 1 and a), a number of proteins were phosphorylated in the basal, nonstimu-
i
M w x 10-3
M W 10-3 ~
-
97.4
- 93 -85 -77
66.2-
-63 -53 -48 - 43
-
4 2.7
0
10 50
p 3 Fig. 1. Representative autoradiograph illustrating CAMP stimulation of rat anterior pituitary protein phosphorylation. Rat anterior pituitaries were dissected and rapidly homogenized in ice-cold 150mM Hepes, 24 mM MgCl, and 0.5 mM EGI’A. Extracts were phosphorylated in vitro in the presence or absence of various concentrations of CAMPaccording to the procedure described in the text. Phosphorylated samples containing equal amounts of protein were electrophoresed on 10%polyacrylamide gels and autoradioqraphs prepared. Lane 1,vehicle; Lane 2 , l O KMCAMP;Lane 3,50 p,M CAMP. Migration of molecular weight standards is provided on the left side of the figure. Cyclic AMP-dependent phosphoproteins are indicated with arrows on the right hand side of the figure. Note that this print of the autoradiographs is slightly overexposed in order that the region of the gel containing the majority of the CAMP-dependentproteins and, in particular, some of the less prominent cAMP-dependent phosphoprotein bands (e.g., 48 and 43 kd) can be highlighted (see also Fig. 3).
lated condition. The most prominent of these had approximate molecular weights (as determined on 10% SDS-polyacrylamide gels) of 137 kd, 114 kd, 85 kd, 77 kd, 63 kd, 53 kd, 39 kd, and 33 kd (see Fig. 1,see also Fig. 3 and Table 11).When the phosphorylated samples were further analyzed using 7.5% polyacrylamide gels to separate higher molecular weight proteins, additional phosphoproteins were revealed. These additional phosphoproteins had apparent molecular weights of 168 kd and 152 kd (see Table 11). The effect of CAMPon the in vitro phosphorylation of anterior pituitary proteins was, examined. Table I summarizes the concentration-dependent effect of CAMPon the amount of “total” phosphorylation within a sample. The incorporation of radiolabelled phosphate into protein was significantly increased during incubation with 10, 50, or 100 pM CAMP. There was essentially no difference in total phosphorylation between 50 and 100 pM CAMP,with the latter providing a maximum stimulation of 118%. Total phosphorylation was also increased following incubation with 1 pM CAMP (38% greater than vehicle), but this increase was not statistically significant. Specific proteins that were phos horylated in a CAMP-dependent manner were identi ied and could be
P
243
ANTERIOR PITUITARY PROTEIN PHOSPHORYLATION
(arbitrary units)
1,438 k 237
1,986 k 158
2,371 i 397*
3,142 k 62*
3,140
+ 245*
‘Anterior pituitary extracts were phosphorylated as described in the text and legend to Figure 1. Autoradiographa were analyzed by densitometry and a peak height obtained for each phosphoprotein. Peak heights weresummed to quantitate the “total” phosphate incorporation into a particular sample. Each value represents the mean i standarderrorofthemean.N=3forallgroupsexceptlO~McAMP, whereN= 2. Results wereanalyzedwithanalysisofvariancefollowedhy Dunnett’stesttocompareeach cAMP concentration to control. *P < 0.05 relative to vehicle.
grouped into two categories. First, CAMPpotentiated the phosphorylation of a oup of proteins that were also phosphorylated in the ve icle condition. Second, several phosphoproteins incorporated radiolabel only in the presence of CAMP. Interestingly, the proteins in this second category were relatively minor phosphoproteins in the overall labelling pattern. The autoradiograph shown in Figure 1 illustrates the phosphoprotein labelling observed on 10% SDS-polyacrylamide gels following incubation with 10 pM (lane 2) and 50 pM CAMP(lane 3) relative t o vehicle (lane 1). It is clearly evident that CAMPstimulated the phosphorylation of several proteins (e.g., 85 kd, 77 kd, 63 kd, and 53 kd), which were also phosphorylated in the vehicle group. Although minimal CAMP stimulation was observed using 1 pM of the cyclic nucleotide (16%increase in 85 and 77 kd phosphorylation, 8% increase in 63 kd phosphorylation, and 12%increase in 53 kd phosphorylation), densitometric analysis following incubation with 50 pM CAMPindicated that the phosphorylation of the 85 kd protein was increased by 61%, the 77 kd protein by 6596, the 63 kd protein by 95%,and the 53 kd protein by 76%. The densitometric quantitation of the dose-related CAMPstimulation of the phosphorylation of these four phosphoproteins is shown in Figure 2A-D. The most dramatic increase in stimulation occurred between 10 and 50 pM CAMP.The CAMPstimulation appeared to plateau near 50 pM CAMP as there was little difference in the magnitude of the stimulatory effect of CAMP between 50 and 100 FM. At 100 p M CAMP,the phosphorylation of the 85 kd protein was increased by 70%, the 77 kd protein by 76%,the 63 kd protein by 10096, and the 53 kd protein by 82%. It was also noted that at CAMP concentrations of 50 pM or greater, the phosphorylation of 39 and 33 kd proteins was potentiated. At lower concentrations of CAMP,the phosphorylation of these proteins was identical to that in the vehicle condition (densitometric d.ata not shown). Also provided in Figure 2 are results illustrating the specificity of CAMP stimulation. As shown in Figure
!r
CONDITION VEHICLE
cAMP POTENTIATED CAMP-DEPENDENT
PHOSPHOPROTEIN MOLECULAR WEIGHT (X lo-.’) 168, 152, 137, 114, 85, 77, 63, 53, 39, 33 85, 77, 63, 53, 39, 33 124, 93, 48, 43
2E-F, the phosphorylation of the 114 and 152 kd proteins was not enhanced in the presence of CAMP.In contrast, the phosphorylation of these proteins may actually be inhibited in the presence of CAMP. We also observed four phosphoproteins that were phosphorylated in vitro only in the presence of cAMP (Figs. 1,3, Table 11).In Figure 1, in lanes 2-3 (10 and 50 pM CAMP)93 kd, 48 kd, and 43 kd proteins are visible. Dose-response studies showed that these proteins were reproducibly phosphorylated in the presence of 10 pM and greater concentrations of CAMP.The 93 kd band is faintly labelled and thus is only slightly visible above background in Figure 1. However, the densitometric tracings corresponding to lanes 1 and 3 in Figure 1 are provided in Figure 3. Here, the peaks corresponding t o the 93 kd, 48 kd, and 43 kd proteins are clearly visible in the CAMPcondition. A fourth CAMP-dependent phosphoprotein was seen when phosphorylated samples were electrophoresed on 7.5% polyacrylamide gels to examine higher molecular weight proteins. In addition to the bands phosphorylated in the vehicle condition, a 124 kd protein was phosphorylated in the presence of 10 pM or greater concentrations of CAMP(not shown). As with the other proteins that were phosphorylated only in the presence of CAMP, the 124 kd protein was faintly labelled. These results from these experiments are summarized in Table 11. Cyclic AMP-stimulated phosphoproteins in the rat anterior pituitary had molecular weights of 124 kd, 93 kd, 85 kd, 77 kd, 63 kd, 53 kd, 48 kd, 43 kd, 39 kd, and 33 kd.
We have identified a number of phosphoproteins that apparently act as substrates for a CAMP-dependentprotein kinase in cell free extracts of the rat anterior pituitary. The in vitro phosphorylation of proteins with molecular weights of 85 kd, 77 kd, 63 kd, 53 kd, 39 kd, and 33 kd is potentiated in the presence of CAMP.The phosphorylation of 124 kd, 93 kd, 48 kd, and 43 kd proteins is reliably observed only in the presence of exogenous CAMPand not in the control condition. As noted in the introduction, CAMP-dependent protein phosphorylation has been previously observed in various pituitary cell lines and nonrodent tissues. In a membrane preparation from bovine anterior pituitary, eleven CAMP-dependent phosphoproteins of wideranging molecular weight were seen, but molecular weights of particular substrates were not identified (Le May et al., 1974). In the AtT-20/D16-16 tumor cell line, it was reported that the adenylate cyclase activator, forskolin, stimulated the phosphorylation of two proteins in the 20-25 kd range, as well as 32,40, and 60 kd
244
S.T. CAIN ET AL
B.
C.
D. Ar
*
L
In
11 0
1
1010-6
L
L
!
~
[CAMP] x
Fig. 2. Dose-dependency of CAMP stimulation of individual phosphoprotein substrates. Autoradiographs obtained following electrophoretic separation of phosphorylated anterior pituitary proteins on 10%or 7.5%polyacrylamide gels were analyzed by microdensitometry. Phosphate incorporation is expressed in arbitrary units based on densitometric peak heights above background. Arbitrary units were normalized so that phosphate incorporation could be expressed on the same scale for each protein. Thus the relative incorporation between proteins is not shown in this figure. (A) 85,000 MW phosphorylation; (B) 77,000 MW phosphorylation; (C) 63,000 MW phosphorylation; (D)
0
1
-
10
L 5c
53,000 MW phosphorylation, (E) 114,OOO MW phodphorylation, (F) 152,000 MW phosphorylation Each bar represents the mean = standard error of the mean (SEM) of three determinations CAMPvalue that represents the mean t SEM of two except the 10 ~J.M determinations Results were tested for statistical significance with a n analysis of variance followed by Dunnel t's test to compare each CAMP concentration to the vehicle group Cyclic AMP stimulated the phosphorylation of the 85,77,63, and 53 kd proteins, but not that ofthe 152 and 114 kd proteins *-I)
Cyclic AMP-Dependent Protein PhosphorylaGon in the Rat Anterior Pituitary SCOTT T. CAIN, JAMES CLIFF PRYOR, AND CHARLES B. NEMEROFF Departments of Psychiatry (S.T.C., J.C.P., C.B.N.) and Pharmacology (C.B.N.), Duke University Medical Center, Durham, North Carolina 27710
KEY WORDS
Cyclic AMP, Protein phosphorylation, Anterior pituitary, Protein kinase
ABSTRACT The activation of cyclic adenosine 3'5'-monophosphate (CAMP)-dependent protein kinases has been implicated as an integral mechanism in stimulus-secretion coupling in the anterior pituitary. Therefore, we have investigated phosphorylation of endogenous protein substrates both in the presence and absence of CAMPin cell-free extracts of the rodent anterior pituitary. Specific phosphoprotein substrates in the rat anterior pituitary, which are phosphorylated by a CAMP-dependent protein kinase in vitro, were identified. Cyclic AMP potentiated the phosphorylation of proteins with apparent molecular weights of 85,000, 77,000, 63,000, 53,000, 39,000, and 33,000 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins with apparent molecular weights of 124,000, 93,000, 48,000, and 43,000 were phosphorylated only in the presence of CAMPand not in the basal condition. The results highlight endogenous protein substrates that may potentially be involved in CAMPdependent stimulus-secretion coupling in the anterior pituitary. INTRODUCTION The receptor-coupled stimulation of CAMPformation and the concomitant activation of CAMP-dependentprotein kinases is believed to be an important exocytotic regulatory mechanism in a number of biological systems (for review see Harper, 1988). In particular, considerable evidence supports the notion that activation of CAMP-dependent protein kinases is a crucial intermediary step in secretagogue-induced release of adenohypophyseal hormones from the anterior pituitary. For example, corticotropin-releasing factor (CRF), the primary physiological regulator of adrenocorticotropin release, activates adenylate cyclase and thus stimulates CAMPformation in both rodent (Aguilera et al., 1983, 1986; Labrie et al., 1982; Sobel, 1985) and primate (Millan et al., 1987) anterior pituitary cells. The doseresponse of CRF-stimulated CAMP formation and CAMP-dependent protein kinase activity closely parallels CRF-stimulated ACTH release (Litvin et al., 1984). Moreover, other hypothalamic hypophysiotrophic hormones such as vasoactive intestinal peptide (Gourdji et al., 19851,growth hormone-releasing factor (Schettini et al., 19841, and luteinizing hormone-releasing hormone (Labrie et al., 1979) are believed to produce their effects on anterior pituitary tro ic hormone release by CAMP-mediated mechanisms. yclic AMP-responsive phosphoprotein substrates have previously been identified in a clonal GH pituitary cell line (Drust et al., 19821, bovine anterior pituitary land (LeMayet al., 19741,and AtT-20/D16-16 tumor ce 1s (Pasmantier et al., 1986; Rougon et al., 1989). However, in spite of this rich literature, little is known of the identity of the CAMP-dependent phos-
e
7
0 1990 WILEY-LISS,INC.
phoprotein substrates in the rodent anterior pituitary, which, in our laboratory and others, is commonly used as a model system to study the hypothalamic-pituitary axis. We have, therefore, examined CAMP-dependent protein phosphorylation in cell-free extracts of the rat anterior pituitary. Although a previous attempt to identify CAMP-dependent phosphoprotein substrates in vitro in the rat anterior pituitar was not successful (Brattin and Portanova, 1981))we ave now identified a number of specific rat anterior pituitary proteins that are responsive to CAMP. MATERIALS AND METHODS Materials Rats were obtained from the Charles River colony in (specific activity 4-5 X Raleigh, N.C. [Y~~PI-ATP lo3 Ci/mM) was obtained from ICN. Electrophoresis equipment and reagants were from Bio-Rad, CAMPwas obtained from Sigma Chemicals, and all other chemicals were reagant grade. Tissue preparation and in vitro phosphorylation Adult male Sprague-Dawley rats were decapitated and the brains rapidly removed to expose the pituitary glands. The anterior pituitary was then separated from the neurointermediate and posterior lobes and homogenized in 20 volumes of 150 mM HEPES, 24 mM MgC12,
K
Received April 27,1989; accepted October 18,1989 Address reprint requests to S. T. Cain, Department of Psychiatry, Box 3859, Duke University Medical Center, Durham, North Carolina 27710.
242
S.T. CAIN ET AL.
and 0.5 mM EGTA, pH 7.1. In vitro phosphorylation was performed using a minor modification of a standard protocol designed to analyze cyclic nucleotide-dependent phosphorylation of endogenous protein substrates (see, e.g., Rudolph and Krueger, 1979). Aliquots of the tissue homogenate were preincubated a t 30°C. After 40 seconds, CAMPor vehicle was added. Twenty seconds later, the phosphorylation reaction was initiated by the addition of ATP (containing [Y~~PI-ATP, approximately 7 X lo6 cpdsample) (final concentrations: 50 mM HEPES, 8 mM MgCl,, 0.17 mM EGTA, 10 pM ATP with or without 1-100 pM CAMP).After 40 seconds of phosphorylation, an aliquot of the sample was removed for trichloroacetic acid (TCA) precipitation on filter papers and liquid scintillation counting. Phosphorylation was allowed to proceed for 60 seconds before being quenched with sodium dodecyl sulfate (SDS) stop solution (final concentration: 3% SDS, 2% P-mercaptoethanol, 1.7%glycerol, 0.01 M Tris pH 8.9). Electrophoresis, autoradiography and densitometric analysis Phosphorylated samples were boiled for 3 minutes at 80°C prior to electrophoresis. Equal amounts of sample proteins were separated on SDS-polyacrylamide gels according to the method of Krueger et al. (1977). The resolving gel consisted of 10% or 7.5% acrylamide, 0.27% bis-acrylamide, 0.375 M Tris pH 8.9, 0.1% N, N, N’, N’ tetramethylethylenediamine (TEMED), and 0.1% SDS. The stacking gel consisted of 5% acrylamide, 0.13% bis-acrylamide, 0.125 M Tris pH 6.8, 0.1% TEMED, and 0.1% SDS. Polymerization of both stacking and resolving gels was initiated by the addition of 0.15% ammonium persulfate. Electrophoresis buffer, both upper and lower chambers, was 0.05 M Tris pH 8.3, 0.375 Mglycine, and 0.1%SDS. Electrophoresis was run at 25 &gel for approximately 1 hour and then at 35 &gel until the bromphenol blue tracking dye was at the bottom of the gel. The gels were fixed and stained in acetic acid-methanol solution containing coomassie blue, dried under vacuum, and autoradiograms prepared by exposing the dried gels to Kodak NS-5T x-ray film for 14 days. Phosphate incorporation into individual proteins was quantitated by microdensitometric analysis of the autoradiograms. Peak height above background was measured for each phosphoprotein. Peak heights for all phosphoproteins within a sample were cumulated to quantitate the “total” hosphorylation in that sample. Molecular weights of p osphorylated proteins were determined by comparison t,o the migration of proteins of known molecular weight. Statistical analysis Vehicle and CAMP-dosle response groups were subjected to a one-way analysis of variance. If the probability of the F score was less than 0.05, dose-response effects were compared to the control level of phosphorylation using Dunnett’s test. Differences were considered statistically significant if p < .05. RESULTS Rat anterior pituitary extracts were phosphorylated with [y3’P]-ATP. As can be seen in the autoradiograph provided in Figure 1 (lanes 1 and a), a number of proteins were phosphorylated in the basal, nonstimu-
i
M w x 10-3
M W 10-3 ~
-
97.4
- 93 -85 -77
66.2-
-63 -53 -48 - 43
-
4 2.7
0
10 50
p 3 Fig. 1. Representative autoradiograph illustrating CAMP stimulation of rat anterior pituitary protein phosphorylation. Rat anterior pituitaries were dissected and rapidly homogenized in ice-cold 150mM Hepes, 24 mM MgCl, and 0.5 mM EGI’A. Extracts were phosphorylated in vitro in the presence or absence of various concentrations of CAMPaccording to the procedure described in the text. Phosphorylated samples containing equal amounts of protein were electrophoresed on 10%polyacrylamide gels and autoradioqraphs prepared. Lane 1,vehicle; Lane 2 , l O KMCAMP;Lane 3,50 p,M CAMP. Migration of molecular weight standards is provided on the left side of the figure. Cyclic AMP-dependent phosphoproteins are indicated with arrows on the right hand side of the figure. Note that this print of the autoradiographs is slightly overexposed in order that the region of the gel containing the majority of the CAMP-dependentproteins and, in particular, some of the less prominent cAMP-dependent phosphoprotein bands (e.g., 48 and 43 kd) can be highlighted (see also Fig. 3).
lated condition. The most prominent of these had approximate molecular weights (as determined on 10% SDS-polyacrylamide gels) of 137 kd, 114 kd, 85 kd, 77 kd, 63 kd, 53 kd, 39 kd, and 33 kd (see Fig. 1,see also Fig. 3 and Table 11).When the phosphorylated samples were further analyzed using 7.5% polyacrylamide gels to separate higher molecular weight proteins, additional phosphoproteins were revealed. These additional phosphoproteins had apparent molecular weights of 168 kd and 152 kd (see Table 11). The effect of CAMPon the in vitro phosphorylation of anterior pituitary proteins was, examined. Table I summarizes the concentration-dependent effect of CAMPon the amount of “total” phosphorylation within a sample. The incorporation of radiolabelled phosphate into protein was significantly increased during incubation with 10, 50, or 100 pM CAMP. There was essentially no difference in total phosphorylation between 50 and 100 pM CAMP,with the latter providing a maximum stimulation of 118%. Total phosphorylation was also increased following incubation with 1 pM CAMP (38% greater than vehicle), but this increase was not statistically significant. Specific proteins that were phos horylated in a CAMP-dependent manner were identi ied and could be
P
243
ANTERIOR PITUITARY PROTEIN PHOSPHORYLATION
(arbitrary units)
1,438 k 237
1,986 k 158
2,371 i 397*
3,142 k 62*
3,140
+ 245*
‘Anterior pituitary extracts were phosphorylated as described in the text and legend to Figure 1. Autoradiographa were analyzed by densitometry and a peak height obtained for each phosphoprotein. Peak heights weresummed to quantitate the “total” phosphate incorporation into a particular sample. Each value represents the mean i standarderrorofthemean.N=3forallgroupsexceptlO~McAMP, whereN= 2. Results wereanalyzedwithanalysisofvariancefollowedhy Dunnett’stesttocompareeach cAMP concentration to control. *P < 0.05 relative to vehicle.
grouped into two categories. First, CAMPpotentiated the phosphorylation of a oup of proteins that were also phosphorylated in the ve icle condition. Second, several phosphoproteins incorporated radiolabel only in the presence of CAMP. Interestingly, the proteins in this second category were relatively minor phosphoproteins in the overall labelling pattern. The autoradiograph shown in Figure 1 illustrates the phosphoprotein labelling observed on 10% SDS-polyacrylamide gels following incubation with 10 pM (lane 2) and 50 pM CAMP(lane 3) relative t o vehicle (lane 1). It is clearly evident that CAMPstimulated the phosphorylation of several proteins (e.g., 85 kd, 77 kd, 63 kd, and 53 kd), which were also phosphorylated in the vehicle group. Although minimal CAMP stimulation was observed using 1 pM of the cyclic nucleotide (16%increase in 85 and 77 kd phosphorylation, 8% increase in 63 kd phosphorylation, and 12%increase in 53 kd phosphorylation), densitometric analysis following incubation with 50 pM CAMPindicated that the phosphorylation of the 85 kd protein was increased by 61%, the 77 kd protein by 6596, the 63 kd protein by 95%,and the 53 kd protein by 76%. The densitometric quantitation of the dose-related CAMPstimulation of the phosphorylation of these four phosphoproteins is shown in Figure 2A-D. The most dramatic increase in stimulation occurred between 10 and 50 pM CAMP.The CAMPstimulation appeared to plateau near 50 pM CAMP as there was little difference in the magnitude of the stimulatory effect of CAMP between 50 and 100 FM. At 100 p M CAMP,the phosphorylation of the 85 kd protein was increased by 70%, the 77 kd protein by 76%,the 63 kd protein by 10096, and the 53 kd protein by 82%. It was also noted that at CAMP concentrations of 50 pM or greater, the phosphorylation of 39 and 33 kd proteins was potentiated. At lower concentrations of CAMP,the phosphorylation of these proteins was identical to that in the vehicle condition (densitometric d.ata not shown). Also provided in Figure 2 are results illustrating the specificity of CAMP stimulation. As shown in Figure
!r
CONDITION VEHICLE
cAMP POTENTIATED CAMP-DEPENDENT
PHOSPHOPROTEIN MOLECULAR WEIGHT (X lo-.’) 168, 152, 137, 114, 85, 77, 63, 53, 39, 33 85, 77, 63, 53, 39, 33 124, 93, 48, 43
2E-F, the phosphorylation of the 114 and 152 kd proteins was not enhanced in the presence of CAMP.In contrast, the phosphorylation of these proteins may actually be inhibited in the presence of CAMP. We also observed four phosphoproteins that were phosphorylated in vitro only in the presence of cAMP (Figs. 1,3, Table 11).In Figure 1, in lanes 2-3 (10 and 50 pM CAMP)93 kd, 48 kd, and 43 kd proteins are visible. Dose-response studies showed that these proteins were reproducibly phosphorylated in the presence of 10 pM and greater concentrations of CAMP.The 93 kd band is faintly labelled and thus is only slightly visible above background in Figure 1. However, the densitometric tracings corresponding to lanes 1 and 3 in Figure 1 are provided in Figure 3. Here, the peaks corresponding t o the 93 kd, 48 kd, and 43 kd proteins are clearly visible in the CAMPcondition. A fourth CAMP-dependent phosphoprotein was seen when phosphorylated samples were electrophoresed on 7.5% polyacrylamide gels to examine higher molecular weight proteins. In addition to the bands phosphorylated in the vehicle condition, a 124 kd protein was phosphorylated in the presence of 10 pM or greater concentrations of CAMP(not shown). As with the other proteins that were phosphorylated only in the presence of CAMP, the 124 kd protein was faintly labelled. These results from these experiments are summarized in Table 11. Cyclic AMP-stimulated phosphoproteins in the rat anterior pituitary had molecular weights of 124 kd, 93 kd, 85 kd, 77 kd, 63 kd, 53 kd, 48 kd, 43 kd, 39 kd, and 33 kd.
We have identified a number of phosphoproteins that apparently act as substrates for a CAMP-dependentprotein kinase in cell free extracts of the rat anterior pituitary. The in vitro phosphorylation of proteins with molecular weights of 85 kd, 77 kd, 63 kd, 53 kd, 39 kd, and 33 kd is potentiated in the presence of CAMP.The phosphorylation of 124 kd, 93 kd, 48 kd, and 43 kd proteins is reliably observed only in the presence of exogenous CAMPand not in the control condition. As noted in the introduction, CAMP-dependent protein phosphorylation has been previously observed in various pituitary cell lines and nonrodent tissues. In a membrane preparation from bovine anterior pituitary, eleven CAMP-dependent phosphoproteins of wideranging molecular weight were seen, but molecular weights of particular substrates were not identified (Le May et al., 1974). In the AtT-20/D16-16 tumor cell line, it was reported that the adenylate cyclase activator, forskolin, stimulated the phosphorylation of two proteins in the 20-25 kd range, as well as 32,40, and 60 kd
244
S.T. CAIN ET AL
B.
C.
D. Ar
*
L
In
11 0
1
1010-6
L
L
!
~
[CAMP] x
Fig. 2. Dose-dependency of CAMP stimulation of individual phosphoprotein substrates. Autoradiographs obtained following electrophoretic separation of phosphorylated anterior pituitary proteins on 10%or 7.5%polyacrylamide gels were analyzed by microdensitometry. Phosphate incorporation is expressed in arbitrary units based on densitometric peak heights above background. Arbitrary units were normalized so that phosphate incorporation could be expressed on the same scale for each protein. Thus the relative incorporation between proteins is not shown in this figure. (A) 85,000 MW phosphorylation; (B) 77,000 MW phosphorylation; (C) 63,000 MW phosphorylation; (D)
0
1
-
10
L 5c
53,000 MW phosphorylation, (E) 114,OOO MW phodphorylation, (F) 152,000 MW phosphorylation Each bar represents the mean = standard error of the mean (SEM) of three determinations CAMPvalue that represents the mean t SEM of two except the 10 ~J.M determinations Results were tested for statistical significance with a n analysis of variance followed by Dunnel t's test to compare each CAMP concentration to the vehicle group Cyclic AMP stimulated the phosphorylation of the 85,77,63, and 53 kd proteins, but not that ofthe 152 and 114 kd proteins *-I)
