Spatiotemporally regulated protein kinase A activity is a critical regulator of growth factor-stimulated extracellular signal-regulated kinase signaling in PC12 cells

Abstract

PC12 cells exhibit precise temporal control of growth factor signaling in which stimulation with epidermal growth factor (EGF) leads to transient extracellular signal-regulated kinase (ERK) activity and cell proliferation, whereas nerve growth factor (NGF) stimulation leads to sustained ERK activity and differentiation. While cyclic AMP (cAMP)-mediated signaling has been shown to be important in conferring the sustained ERK activity achieved by NGF, little is known about the regulation of cAMP and cAMP-dependent protein kinase (PKA) in these cells. Using fluorescence resonance energy transfer (FRET)-based biosensors localized to discrete subcellular locations, we showed that both NGF and EGF potently activate PKA at the plasma membrane, although they generate temporally distinct activity patterns. We further show that both stimuli fail to induce cytosolic PKA activity and identify phosphodiesterase 3 (PDE3) as a critical regulator in maintaining this spatial compartmentalization. Importantly, inhibition of PDE3, and thus perturbation of the spatiotemporal regulation of PKA activity, dramatically increases the duration of EGF-stimulated nuclear ERK activity in a PKA-dependent manner. Together, these findings identify EGF and NGF as potent activators of PKA activity specifically at the plasma membrane and reveal a novel regulatory mechanism contributing to the growth factor signaling specificity achieved by NGF and EGF in PC12 cells.

Fig. 1.

GF-induced PKA activity is spatiotemporally regulated in PC12 cells. (A) Schematic representation of AKAR4-Kras and AKAR4-NES. (B) PC12 cells expressing AKAR4-NES (n = 3) or AKAR4-Kras (n = 6) were treated with Fsk (50 μM) plus IBMX (100 μM). Y/C, yellow/cyan emission ratio. (C) Time courses depicting the NGF (200 ng/ml)-induced responses of AKAR4-Kras (n = 5) and AKAR4-NES (n = 5). (D) YFP images and ratiometric images of AKAR4-Kras (top) and AKAR4-NES (bottom) before NGF addition (0 min) and at 5 min and 12.5 min after NGF addition. (E) Time courses depicting the EGF (100 ng/ml)-induced responses of AKAR4-Kras (n = 6) and AKAR4-NES (n = 3). (F) YFP images and ratiometric images of AKAR4-Kras (top) and AKAR4-NES (bottom) before EGF addition (0 min) and at 5 min and 12.5 min after EGF addition. (G) Bar graph depicting the AKAR4-Kras responses at 2.5, 5, 10, and 12.5 min following NGF (n = 11) or EGF (n = 12) treatment. n.s, no statistically significant difference between indicated treatments; **, P ≤ 0.01; ***, P ≤ 0.005. All data are shown as means ± SEMs.

Fig. 2.

PDE3, but not PDE4, regulates both PM and cytosolic PKA activity in PC12 cells. (A) PC12 cells expressing AKAR4-Kras were treated with 100 μM IBMX (n = 4), 10 μM milrinone (Mil) (n = 5), or 1 μM rolipram (Roli) (open circles; n = 5), and PKA activity was monitored. (B) Cytosolic PKA activity was detected in cells expressing AKAR4-NES treated with 100 μM IBMX (n = 4), 10 μM Mil (n = 5), or 1 μM Roli (n = 6). (C) Responses of AKAR4-Kras and AKAR4-NES induced by IBMX (Kras [n = 13], NES [n = 9]), Mil (Kras [n = 16], NES [n = 7]), and Roli (Kras [n = 9], NES [n = 15]). (D) Bar graph depicting the cAMP concentration ([cAMP]) induced by various treatments, as determined by Epac1-camps [50 nM Fsk (n = 11), 100 μM IBMX (n = 8), 10 μM Mil (n = 15), 1 μM Roli (n = 11)]. (E) PC12 cells were separated into cytosolic (C) and membrane (M) fractions by centrifugation, and each fraction was separated by SDS-PAGE. Immunoblot analysis (top) is representative of one fractionation, and quantification of the relative expression of PDE3B in each fraction (bottom) is representative of four independent fractionation experiments and subsequent immunoblot analysis. Tubulin serves as a control for clean separation of the membrane and cytosolic fractions. All data are presented as means ± SEMs; “n.s.” indicates no statistically significant difference between indicated treatments. **, P < 0.01; ***, P < 1E−05.

Fig. 3.

GFs activate cytosolic PKA when PDE3 is partially inhibited. (A) Bar graph depicting the negligible AKAR4-NES response induced by GF alone (white bars; NGF [n = 4] and EGF [n = 12]) and a subsaturating dose of milrinone (5 μM) (gray bars; n = 10). In contrast, when cells are simultaneously treated with GF and 5 μM milrinone (black bars; NGF [n = 12] and EGF [n = 6]), an increase in cytosolic PKA activity is observed. (B) Time course of PC12 cells expressing AKAR4-NES treated with NGF alone (n = 5) or NGF plus 5 μM milrinone (submil n = 3). (C) Time course depicting the AKAR4-NES response of cells treated with EGF alone (n = 3) or EGF plus 5 μM milrinone (n = 3). (D) Bar graph depicting the cAMP concentration induced by various treatments, as determined by the response of Epac1-camps (NGF [n = 8], EGF [n = 7], 5 μM Mil [n = 15], NGF plus 5 μM Mil [n = 14], and EGF plus 5 μM Mil [n = 10]). (E) A submaximal dose of rolipram (0.5 μM) does not activate cytosolic PKA in the absence (gray bars; n = 5) or presence (black bars; NGF [n = 11] and EGF [n = 9]) of GF. (F) The EGF-induced PKA activity at the plasma membrane (n = 12) is sustained in the presence of PDE3 inhibition with milrinone (10 μM) (n = 8) but not in the presence of PDE4 inhibition with rolipram (1 μM) (n = 6). All data are shown as means ± SEMs. *, P < 0.05.

Fig. 4.

cAMP-mediated signaling regulates ERK activity in PC12 cells. (A) Bar graph depicting the time to reach half-maximal activation [(t(1/2)] of EKARcyto in response to various treatments (NGF alone [n = 12], NGF after H89 pretreatment [n = 14], NGF in the presence of PKIα expression [n = 5], EGF alone [n = 23], EGF after H89 pretreatment [n = 13], and EGF in the presence of PKIα expression [n = 5]). (B) Time courses of PC12 cells expressing EKAR_cyto treated with NGF (Cntl; n = 8), NGF after 10 μM H89 pretreatment (n = 7), or NGF in the presence of PKIα expression (n = 5). (C) Time courses depicting the EKAR_cyto response to EGF (cntl; n = 8), EGF after H89 pretreatment (n = 4), and EGF in the presence of PKIα expression (n = 3). (D and E) NGF (D)- and EGF (E)-induced time courses of ERK activity in cells in which PKA anchoring was disrupted via pretreatment with Ht31 (1 μM). (F) Bar graph depicting the time to reach half-maximal activation of EKAR_cyto in response to various treatments (NGF alone [n = 12], NGF after KH7 pretreatment [n = 14], EGF alone [n = 23], and EGF after KH7 pretreatment [n = 13]). (G) Time courses of PC12 cells expressing EKAR_cyto treated with NGF (Cntl; n = 8) or NGF after 30 μM KH7 pretreatment (n = 7). (H) Time courses depicting the EKAR_cyto response to EGF (Cntl; n = 8) and EGF after KH7 pretreatment (n = 7). (I) PC12 cells were pretreated with vehicle, 30 μM KH7 (K), or 10 μM H89 (H) for 20 min and subsequently treated with NGF (N) or EGF (E) for 5 min or 15 min. Control cells (Cntl) were treated with vehicle only. Immunoblots of p-ERK1/2 and tubulin (loading control) represent a single experiment. (J) Quantifications of p-ERK1/2 over tubulin for the NGF-treated samples are plotted and normalized to the vehicle-treated sample at each time point (n = 3 to 5 independent experiments). (K) Quantifications of p-ERK1/2 over tubulin for the EGF-treated samples are plotted and normalized to the vehicle-treated sample at each time point (n = 3 to 5 independent experiments). All imaging and immunoblot data are shown as means ± SEMs. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.d., not detected.

Fig. 5.

PDE3, but not PDE4, regulates ERK activity in PC12 cells. (A) Bar graph depicting the t_1/2s of various treatments (NGF alone [n = 12], NGF after milrinone [M] pretreatment [n = 14], NGF after pretreatment with 1 μM rolipram [R] [n = 7], EGF alone [n = 23], EGF after milrinone pretreatment [n = 12], and EGF after rolipram pretreatment [n = 16]). (B) Time courses depicting the EKAR_cyto response from cells treated with NGF (Cntl; n = 8) and NGF after pretreatment with 10 μM milrinone (Mil; n = 8). (C) Time courses depicting the EKAR_cyto response of cells treated with EGF (Cntl; n = 8) and EGF after pretreatment with milrinone (n = 6). (D) PC12 cells were pretreated with vehicle or 10 μM milrinone (M) for 20 min and subsequently treated with NGF or EGF for 2.5 min, 5 min, or 15 min. Control cells (Cntl) were treated with vehicle only. Immunoblots of p-ERK1/2 and tubulin represent a single experiment. (E) Quantifications of p-ERK1/2 over tubulin for the NGF-treated samples are plotted and normalized to the vehicle-treated sample at each time point (n = 3 independent experiments). (F) Quantifications of p-ERK1/2 over tubulin for the EGF-treated samples are plotted and normalized to the vehicle-treated sample at each time point (n = 3 independent experiments). All imaging and immunoblot data are shown as means ± SEMs. *, P < 0.05; **, P < 0.01; n.d., not detected.

Fig. 6.

PDE3 inhibition extends the duration of EGF-induced nuclear ERK in a PKA-dependent manner. All experiments are done in PC12 cells expressing EKAR_nuclear. (A) YFP direct (left) and ratiometric images depicting differential GF-induced nuclear ERK activities in the presence and absence of PDE3 inhibitor (Mil) and/or PKA inhibitor (H89). (B) Graphical representation of the time needed for nuclear ERK activity to reduce to 50% [t_1/2(Rev)]. (C) NGF-induced nuclear ERK activity (n = 9) is more sustained than EGF-induced nuclear ERK activity (n = 10). (D) Time course of NGF-induced nuclear ERK activity in the presence of H89 (n = 8) and PKIα (n = 5) expression. (E) tmAC activation with increasing doses of Fsk extends the duration of EGF-stimulated ERK activity in the nucleus (0.05 μM Fsk, n = 8; 0.5 μM Fsk, n = 9; 5.0 μM Fsk, n = 10; 50 μM Fsk, n = 7. (F) Bar graph depicting the increase in reversal of nuclear ERK activity [t_1/2(Rev)] following various treatments (EGF plus the following doses of Fsk: 0.05 μM, n = 16; 0.5 μM, n = 17; 5.0 μM, n = 10; 50 μM Fsk, n = 7). (G) PDE3 inhibition with 10 μM Mil (n = 7) extends the duration of EGF-stimulated ERK activity. (H and I) 100 μM IBMX increases the duration of EGF-stimulated nuclear ERK activity (n = 8) (H), while 1 μM Roli does not (n = 7) (I). (J) In the presence of milrinone and inhibition of PKA through H89 pretreatment (n = 8) or PKIα expression (n = 5), there is no increase in the duration of EGF-induced nuclear ERK activity. All data are shown as means ± SEMs. δδ, P < 0.01 compared to EGF. **, P ≤ 0.01 compared to NGF.

Fig. 7.

The duration of nuclear ERK activity does not precisely correlate with neurite outgrowth. PC12 cells expressing a plasma membrane-targeted YFP were treated for 3 days as indicated, and the number of cells expressing neurites (defined as a cellular extension longer than the length of the cell body) was quantified. n = 25 to 105 cells per treatment. Representative images for each treatment are shown.