Arthur S. Alberts

Transcriptional attenuation following cAMP induction requires PP-1-mediated dephosphorylation of CREB

We have examined the mechanism by which the transcriptional activity of the cAMP-responsive factor CREB is attenuated following induction with forskolin. Metabolic labeling studies reveal that, after an initial burst of phosphorylation in response to cAMP, CREB is dephosphorylated and transcription of the cAMP-responsive somatostatin gene is correspondingly reduced. The phosphatase inhibitor 1 protein and okadaic acid both prevented the dephosphorylation of CREB at Ser-133 in PC12 cells and also augmented the transcriptional response to cAMP. Of the four Ser/Thr phosphatases described to date, only PP-1 appears to be similarly inhibited by these agents. As PP-1 specifically dephosphorylates CREB at Ser-133 and inhibits cAMP-dependent transcription, we propose that this phosphatase is the major regulator of CREB activity in cAMP-responsive cells.

Expression of a peptide inhibitor of protein phosphatase 1 increases phosphorylation and activity of CREB in NIH 3T3 fibroblasts.

We have examined the activity and phosphorylation state of the cyclic AMP (cAMP) response element binding factor (CREB) in intact NIH 3T3 cells following microinjection of expression plasmids encoding regulatory proteins of type 1 (PP1) and 2A (PP2A) serine/threonine-specific protein phosphatases. Changes in CREB phosphorylation in the injected cells were monitored by indirect immunofluorescence using an affinity-purified antiserum (Ab5322) which specifically recognizes CREB phosphorylated at Ser-133, and changes in transcriptional activity of CREB were monitored by expression of a reporter gene regulated by cAMP. cAMP-stimulated phosphorylation in NIH 3T3 cells is normally transient, and as expected, after stimulation of cells with cell-permeable cAMP analogs, the level of phosphorylated CREB was found to initially increase and then return to a basal level within 4 h. Microinjection of an expression vector encoding a constitutively active form of inhibitor 1 (I-1), a PP1-specific inhibitor, by itself resulted in an apparent increase in phosphorylated CREB in unstimulated cells. Moreover, injection of the I-1 vector resulted in the prolonged appearance of phosphorylated CREB in cells after cAMP stimulation. In contrast, injection of a plasmid encoding simian virus 40 small t antigen, which interacts with PP2A to inhibit its activity towards several phosphoprotein substrates, had no effect on the phosphorylation state of CREB in stimulated or unstimulated NIH 3T3 cells. Consistent with these results, injection of the I-1 expression vector activated expression from a coinjected CRE-lacZ reporter plasmid, indicating that the increased phosphorylation of CREB also activated its transcriptional activity. These results provide further evidence for a role of a PP1 as the primary protein (Ser/Thr) phosphatase regulating the dephosphorylation of Ser-133 and thereby limiting the transcriptional activity of CREB.

Signal transduction through the cAMP-dependent protein kinase

Temporal cellular events responsible for hormonal activation of responses mediated by the cAMP-dependent protein kinase (PKA) have been studied in living cells. By selectively perturbing molecular function of Gs, the catalytic subunit of PKA (C), or the nuclear factor CREB, in cells through microinjection of inhibitory agents specific for these molecules or activated forms of these molecules, we have obtained evidence for a requirement for the function of each of these molecules in the hormonal stimulation of cAMP-regulated genes. Moreover, by introducing fluorescently labeled PKA subunits into these cells as molecular tracers, or by immunofluorescence of C subunit, we have observed biological translocation of C subunit from the cytoplasm to the nucleus during transcriptional activation and a quenching of this by the inhibitor molecule, PKI. The implications of these cellular and molecular events in the signal transduction of hormonal responses are discussed.

Protein phosphatase 2A potentiates activity of promoters containing AP-1-binding elements.

The involvement of serine/threonine protein phosphatases in signaling pathways which modulate the activity of the transcription factor AP-1 was examined. Purified protein phosphatase types 1 (PP1) and 2A (PP2A) were microinjected into cell lines containing stably transfected lacZ marker genes under the control of an enhancer recognized by AP-1. Microinjection of PP2A potentiated serum-stimulated beta-galactosidase expression from the AP-1-regulated promoter. Similarly, transient expression of the PP2A catalytic subunit with c-Jun resulted in a synergistic transactivation of an AP-1-regulated reporter gene. PP2A, but not PP1, potentiated serum-induced c-Jun expression, which has been previously shown to be autoregulated by AP-1 itself. Consistent with these results, PP2A dephosphorylated c-Jun on negative regulatory sites in vitro, suggesting one possible direct mechanism for the effects of PP2A on AP-1 activity. Microinjection of PP2A had no effect on cyclic AMP (cAMP)-induced expression of a reporter gene containing a cAMP-regulated promoter, while PP1 injection abolished cAMP-induced gene expression. Taken together, these results suggest a specific role for PP2A in signal transduction pathways that regulate AP-1 activity and c-Jun expression.

Kinetic studies of mutant human adenylosuccinase.

Residual adenylosuccinase activity was studied in cultured lymphoblasts from a pair of siblings with infantile autism who have been previously shown to have a deficiency of the enzyme. The rates and distribution of de novo purine synthesis by intact cells were nearly normal. There was no evidence of inhibitory activity in the lysates of the mutant cells. The optimal pH was indistinguishable from that in control cells. The apparent Km in the two mutant cells lines is not significantly different from normal, but the mutants displayed markedly decreased maximum steady-state velocities. Residual activities in mutant cells show decreased thermal stability, suggesting that there is a structural mutation of the adenylosuccinase in the mutant cells.

Positive and Negative Regulation of Cell Cycle Progression by Serine/Threonine Protein Phosphatases

Reversible protein phosphorylation events play a key role in intracellular signal transduction pathways that regulate gene expression and cell proliferation. To analyze the involvement of protein phosphatases in these processes, we applied two different approaches. First, we used okadaic acid, a specific inhibitor of the two major serine/threonine protein phosphatases, type 1 (PP-1) and type 2A (PP-2A).1–3 Second, we microinjected subunits of PP-2A into living cells and analyzed the effects on gene expression.

9 STUDIES OF MUTANT HUMAN ADENYLOSUCCINATE LYASE

Residual adenylosuccinate lyase activity was studied in cultured lymphoblasts from a pair of siblings who have mental retardation and autism, and who have been previously shown to exhibit a deficiency of the enzyme. Utilization of formate by intact cells showed de novo synthesis in the mutant lymphoblasts to be similar to normal, consistent with a partial deficiency of this enzyme as previously reported. The steady-state kinetics and thermal stability of adenylosuccinate lyase were examined in the lymphoblast lysates. The pH-activity curves were nearly identical for the mutant and normal cells, with an optimum in the region of pH 7.8. While the substrate affinity was not distinguishable from normal, there was a significant difference (p < 0.05) In the Vmax of the mutant cells. The lyase from normal cells exhibited an apparent Km of 3.3 ± 0.8 μM and a Vmax of 13.8 ± 0.9 nmol·mg−1·min−1. In the mutant cells, the lyase had an apparent Km of 2.6 ± 0.5 μM and Vmax of 6.7 ± 1.1 nmol mg−1·min−1. There was a significant difference between the normal and mutant cell lyases with regard to thermal stability. At 37°C, the half-time for inactivation was 22.2 hours for the normal cells and 15.7 hours for the mutant cell lysates, and at 60°C the half-times were 5.0 and 0.49 minutes for normal and mutant cell lysates, respectively. There was no evidence of inhibitory activity in the lysates of the mutant cells. The results are consistent with a structural mutation in the adenylosuccinate lyase gene of the affected individuals.