甲硫氨酸脑啡肽对猴免疫缺陷病毒早期感染引起CEM x1 74细胞凋亡的可能作用(英文)

探讨短期甲硫氨酸脑啡肽作用对SIV感染CEMx174细胞凋亡的可能作用 .用MTS法测定甲硫氨酸脑啡肽对感染CEMx174细胞存活率的影响 ,流式细胞仪分析SIV诱导细胞凋亡及其甲硫氨酸脑啡肽的作用 ,并测定了cAMP含量、PKA活性和组蛋白磷酸化的水平 .SIV能够显著减少CEMx174细胞数 ,甲硫氨酸脑啡肽可以提高感染细胞的存活率 .AnnexinⅤ结合实验显示 ,1μmol L甲硫氨酸脑啡肽可以增加存活细胞的比率 ,减少凋亡细胞数 .甲硫氨酸脑啡肽降低正常细胞和感染细胞cAMP的含量和PKA活性 ,但是在感染组较为明显 .在感染的情况下 ,磷酸化的组蛋白增加 ,甲硫氨酸脑啡肽可以减少其含量 .甲硫氨酸脑啡肽的作用可以被纳酪酮所拮抗 .研究结果提示 ,甲硫氨酸脑啡肽对SIV感染引起早期细胞凋亡的作用涉及cAMP PKA信号传递过程     

Involvement of caspase-3 pathway in anti-apoptotic action of methionine enkephalin on CEM x 174 cells in prolonged infection with simian immunodeficiency virus in vitro

The roles of methionine enkephalin, as an immunomodulator, on immunodeficiency virus-induced apoptosis of lymphocytes during prolonged infection are still unclear. In the present study, we evaluated the effects of methionine enkephalin on the viability, the profile of cell cycle and apoptosis, as well as the expression of apoptosis-related genes in CEM x 174 cells infected with simian immunodeficiency virus for 72 h. Our data demonstrated that methionine enkephalin maintains the viability of cells during the period of prolonged infection. Following co-incubation with the virus, CEM x 174 cells were arrested at S phase, with increased mortality as a result of apoptosis. Methionine enkephalin could abolish virus-induced over-expression of caspase-3. Taken together all findings, we conclude that methionine enkephalin may maintain the viability of SIV-infected cells via suppressing the expression of caspase-3, which may have clinical implications in opioid peptide therapy for AIDS.     

Protein kinase A (PKA) isoform RIIbeta mediates the synergistic killing effect of cAMP and glucocorticoid in acute lymphoblastic leukemia cells

Protein kinase A (PKA) or cAMP-dependent protein kinase (cAPK) mediates the synergistic effects of cAMP- and glucocorticoid (GC)-induced apoptosis in lymphoid cells. Using two human acute lymphoblastic leukemia cell (CEM) clones with respective GC-sensitive and GC-resistant phenotypes, we discovered that the PKA regulatory subunit isoform RII(beta) is preferentially expressed in the GC-sensitive clone C7-14 cells, whereas other intracellular cAMP receptors, including the exchange proteins directly activated by cAMP (Epac), are expressed at similar levels in both GC-sensitive and GC-resistant clones. High RII(beta) expression level in C7-14 cells is associated with elevated total PKA cellular activity and cAMP sensitivity, which consequently lead to an increased basal PKA activity. cAMP analogs that selectively activate type II PKA recapitulate the effects of forskolin of promoting apoptosis and antagonizing AKT/PKB activity in both GC-sensitive and GC-resistant clones, whereas type I PKA-selective agonists do not. Furthermore, down-regulation of RII(beta) leads to increased AKT/PKB activation and enhanced GC resistance in C7-14 cells. These results demonstrate that PKA RII(beta) is responsible for increased GC sensitivity, critical for cAMP-mediated synergistic cell killing in CEM cells, and may represent a novel therapeutic target for GC-resistant lymphoid malignancy.     

阿片类物质对猴免疫缺陷病毒感染的CEM×174细胞内p53合成的调节作用(英文)

已经证明阿片类物质如吗啡能够刺激猴免疫缺陷病毒 ( SIV)的复制 ,并最终加速细胞死亡 ,但是其机理却研究很少 .为探讨吗啡和甲硫氨酸脑啡肽对细胞内 p53合成的作用 ,用 SIV感染CEM× 1 74细胞 ,同时分析 SIV感染时病理过程的机理 .在 CEM× 1 74细胞感染 SIV后不同时间 ,p53含量逐渐增加 ,但 1 0 -7mol/L的吗啡仅在起始阶段对其有促进作用 .在 SIV感染组加入吗啡或甲硫氨酸脑啡肽进行时间曲线实验时 ,p53含量较低 .加入 1 0 -8～ 1 0 -6mol/L吗啡 8h,正常细胞 p53含量仅有轻微改变 .但在 SIV感染情况下 ,则呈现剂量依赖性的大量增加 .相反 ,1 0 -8- 1 0 -6mol/L甲硫氨酸脑啡肽在 8h时能增加正常细胞 p53合成达 60 % .在 SIV感染时 ,SIV本身能够促进 p53的含量 .尽管各组 p53仍然高于对照组 ,但甲硫氨酸脑啡肽对其不再起作用 .结果提示甲硫氨酸脑啡肽对正常细胞 p53含量有明显影响 ,而吗啡 8h增加 SIV感染细胞的 p53含量可能是其加速爱滋病病理过程的机理之一 .     

Morphine suppresses lymphocyte apoptosis by blocking p53-mediated death signaling

Opiates such as morphine or heroin may promote cell apoptosis and cause dysfunction of immune cells. In simian immunodeficiency virus (SIV)-infected lymphocytic cells, however, morphine may protect the cells from apoptotic lysis and allow the virus to continue to replicate. To further explore this apparently antithetical effect of opiates, we evaluated in the present study the effects of morphine on human lymphocytic CEM x174 cells induced to undergo apoptosis in the presence of actinomycin D. It was found that induction of apoptosis (characterized by DNA laddering) by actinomycin D was accompanied by a stimulation of the expression of active (phosphorylated) form of p53. Pretreatment of the cells with 10nM morphine caused a transient, naloxone-reversible suppression of the appearance of activated p53 and the generation of DNA laddering. Parallel evaluation of the growth of CEM x174 indicated that morphine treatment delays the inception of cell death triggered by actinomycin D. Inasmuch as Bcl-2 suppresses while Bax accelerates apoptosis, treatment of cells with morphine reduced the expression of Bax and enhanced the expression of Bcl-2. Taken together, morphine, through binding at the opioid receptor, may protect lymphocytic cells from apoptotic lysis if cell death is initiated by apoptosis-inducing agents such as human immunodeficiency virus (HIV), SIV or actinomycin D.     

Dopamine and cyclic AMP-regulated phosphoprotein-32 phosphorylation pattern in cocaine and morphine-sensitized rats

This study reports some of the modifications in dopaminergic signalling that accompany cocaine and morphine behavioural sensitization. Cocaine-sensitized rats showed increased phosphorylation of dopamine- and cyclic AMP-regulated phosphoprotein Mr 32 kDa (DARPP-32) at threonine-75 (Thr75) and decreased DARPP-32 phosphorylation at Thr34, in the caudate-putamen (CPu) and nucleus accumbens (NAc) 7 days after sensitization assessment. Conversely, in morphine-sensitized rats, no apparent modifications in DARPP-32 phosphorylation pattern were observed. Morphine-sensitized rats have increased binding and coupling of micro -opioid receptors and increased dopaminergic transmission in striatal areas and, upon morphine challenge, exhibit dopamine D1 receptor-dependent stereotypies. Thus, the DARPP-32 phosphorylation pattern was studied in morphine-sensitized rats at different times after morphine challenge. Morphine challenge increased levels of phospho-Thr75 DARPP-32 and decreased levels of phospho-Thr34 DARPP-32 in a time-dependent manner in the CPu and NAc. In order to assess whether these modifications were related to modified cyclic AMP-dependent protein kinase (PKA) activity, the phosphorylation levels of two other PKA substrates were examined, the GluR1 and NR1 subunits of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate and NMDA receptors respectively. The phosphorylation levels of GluR1 and NR1 subunits decreased in parallel with those of phospho-Thr-34 DARPP-32, supporting the hypothesis that morphine challenge elicited a decrease in PKA activity in morphine-sensitized rats.     

Cyclic AMP Signaling Reduces Sirtuin 6 Expression in Non-small Cell Lung Cancer Cells by Promoting Ubiquitin-Proteasomal Degradation via Inhibition of the Raf-MEK-ERK (Raf/Mitogen-activated Extracellular Signal-regulated Kinase/Extracellular Signal-regulated Kinase) Pathway

The cAMP signaling system regulates various cellular functions, including metabolism, gene expression, and death. Sirtuin 6 (SIRT6) removes acetyl groups from histones and regulates genomic stability and cell viability. We hypothesized that cAMP modulates SIRT6 activity to regulate apoptosis. Therefore, we examined the effects of cAMP signaling on SIRT6 expression and radiation-induced apoptosis in lung cancer cells. cAMP signaling in H1299 and A549 human non-small cell lung cancer cells was activated via the expression of constitutively active Gα_s plus treatment with prostaglandin E2 (PGE2), isoproterenol, or forskolin. The expression of sirtuins and signaling molecules were analyzed by Western blotting. Activation of cAMP signaling reduced SIRT6 protein expression in lung cancer cells. cAMP signaling increased the ubiquitination of SIRT6 protein and promoted its degradation. Treatment with MG132 and inhibiting PKA with H89 or with a dominant-negative PKA abolished the cAMP-mediated reduction in SIRT6 levels. Treatment with PGE2 inhibited c-Raf activation by increasing inhibitory phosphorylation at Ser-259 in a PKA-dependent manner, thereby inhibiting downstream MEK-ERK signaling. Inhibiting ERK with inhibitors or with dominant-negative ERKs reduced SIRT6 expression, whereas activation of ERK by constitutively active MEK abolished the SIRT6-depleting effects of PGE2. cAMP signaling also augmented radiation-induced apoptosis in lung cancer cells. This effect was abolished by exogenous expression of SIRT6. It is concluded that cAMP signaling reduces SIRT6 expression by promoting its ubiquitin-proteasome-dependent degradation, a process mediated by the PKA-dependent inhibition of the Raf-MEK-ERK pathway. Reduced SIRT6 expression mediates the augmentation of radiation-induced apoptosis by cAMP signaling in lung cancer cells.     

Simian immunodeficiency virus variants with differential T-cell and macrophage tropism use CCR5 and an unidentified cofactor expressed in CEMx174 cells for efficient entry.

The recent identification of coreceptors that mediate efficient entry of human immunodeficiency virus type 1 (HIV-1) suggests new therapeutic and preventive strategies. We analyzed simian immunodeficiency virus (SIV) entry cofactors to investigate whether the macaque SIV model can be used as an experimental model to evaluate these strategies. Similar to primary HIV-1 isolates, a well-characterized molecular clone, SIVmac239, which replicates poorly but efficiently enters into rhesus alveolar macrophages and an envelope variant, SIVmac239/316Env, with an approximately 1,000-fold-higher replicative capacity in macrophages used the beta-chemokine receptor CCR5 for efficient entry. The transmembrane portion of 316Env allowed low-level entry into cells expressing CCR1, CCR2B, and CCR3. A single amino acid substitution in the V3 loop of SIVmac239/316Env, 321P-->S, impaired the ability to enter into the T-B hybrid cell line CEMx174 but had relatively little effect on entry into primary cells and HOS.CD4 cells expressing CCR5. Although CEMx174 cells do not express CCR5, most SIVmac variants entered this hybrid cell line efficiently but did not enter the parental T-cell line CEM. It seems likely that CEMx174 cells express an as-yet-unidentified, perhaps B-cell-derived cofactor which allows efficient entry of SIVmac.     

Transcription factor AP-2 activity is modulated by protein kinase A-mediated phosphorylation

We recently reported that APOE promoter activity is stimulated by cAMP, this effect being mediated by factor AP-2 [Garcia et al. (1996) J. Neurosci. 16, 7550-7556]. Here, we study whether cAMP-induced phosphorylation modulates the activity of AP-2. Recombinant AP-2 was phosphorylated in vitro by protein kinase A (PKA) at Ser239. Mutation of Ser239 to Ala abolished in vitro phosphorylation of AP-2 by PKA, but not the DNA binding activity of AP-2. Cotransfection studies showed that PKA stimulated the effect of AP-2 on the APOE promoter, but not that of the S239A mutant. Therefore, cAMP may modulate AP-2 activity by PKA-induced phosphorylation of this factor.     

Constitutive steroidogenesis in ovine large luteal cells may be mediated by tonically active protein kinase A

The mechanisms responsible for the increased basal rates of progesterone secretion from large steroidogenic luteal cells (LLC) relative to small steroidogenic luteal cells (SLC) have not been clearly defined. To determine if protein kinase A (PKA) is tonically active in LLC, the adenylate cyclase activator forskolin and a specific PKA inhibitor (PKI) were utilized in a 2 x 2 factorial treatment with each steroidogenic cell type. Progesterone and cAMP production were quantified after the different treatments. In addition, the effects of the treatments on the concentrations and relative phosphorylation status of the steroidogenic acute regulatory (STAR) protein in the two cell types were determined as a measure of PKA activity. Treatment with PKI blocked forskolin-induced increases in progesterone secretion by SLC without affecting the production of cAMP. The treatment of LLC with PKI significantly decreased basal progesterone secretion in the presence or absence of forskolin, indicating that the high level of steroidogenesis in this cell type requires PKA activity. There were no differences in the steady-state concentrations of STAR protein in either cell type after treatment. However, the percentage of relative STAR phosphorylation was higher in the LLC than in SLC, and PKI treatment significantly decreased the phosphorylation of STAR in the LLC. The relative phosphorylation status of STAR and the concentrations of progesterone in the media were significantly correlated with the treatments in both cell types. The amount of progesterone secreted per picogram of cAMP was higher in the LLC than in the SLC, and this was accompanied by a significant increase in the ratio of relative STAR phosphorylation to the steady-state concentration of STAR protein. These data are compatible with the theory that LLC are constitutively steroidogenic, partly because they have tonically active PKA. In addition, the phosphorylation of STAR appears to be a primary activity of PKA in both types of ovine steroidogenic luteal cells.     

Neutralizing Antibodies in Sera from Macaques Immunized with Attenuated Simian Immunodeficiency Virus

Infection with attenuated simian immunodeficiency virus (SIV) in rhesus macaques has been shown to raise antibodies capable of neutralizing an animal challenge stock of primary SIVmac251 in CEMx174 cells that correlate with resistance to infection after experimental challenge with this virulent virus (M. S. Wyand, K. H. Manson, M. Garcia-Moll, D. C. Montefiori, and R. C. Desrosiers, J. Virol. 70:3724–3733, 1996). Here we show that these neutralizing antibodies are not detected in human and rhesus peripheral blood mononuclear cells (PBMC). In addition, neutralization of primary SIVmac251 in human and rhesus PBMC was rarely detected with plasma samples from a similar group of animals that had been infected either with SIVmac239Δnef for 1.5 years or with SIVmac239Δ3 for 3.2 years, although low-level neutralization was detected in CEMx174 cells. Potent neutralization was detected in CEMx174 cells when the latter plasma samples were assessed with laboratory-adapted SIVmac251. In contrast to primary SIVmac251, laboratory-adapted SIVmac251 did not replicate in human and rhesus PBMC despite its ability to utilize CCR5, Bonzo/STRL33, and BOB/gpr15 as coreceptors for virus entry. These results illustrate the importance of virus passage history and the choice of indicator cells for making assessments of neutralizing antibodies to lentiviruses such as SIV. They also demonstrate that primary SIVmac251 is less sensitive to neutralization in human and rhesus PBMC than it is in established cell lines. Results obtained in PBMC did not support a role for neutralizing antibodies as a mechanism of protection in animals immunized with attenuated SIV and challenged with primary SIVmac251.     

Viral determinants of simian immunodeficiency virus (SIV) virulence in rhesus macaques assessed by using attenuated and pathogenic molecular clones of SIVmac.

To identify viral determinants of simian immunodeficiency virus (SIV) virulence, two pairs of reciprocal recombinants constructed from a pathogenic (SIVmac239) and a nonpathogenic (SIVmac1A11) molecular clone of SIV were tested in rhesus macaques. A large 6.2-kb fragment containing gag, pol, env, and the regulatory genes from each of the cloned (parental) viruses was exchanged to produce one pair of recombinant viruses (designated SIVmac1A11/239gag-env/1A11 and SIVmac239/1A11gag-env/239 to indicate the genetic origins of the 5'/internal/3' regions, respectively, of the virus). A smaller 1.4-kb fragment containing the external env domain of each of the parental viruses was exchanged to create the second pair (SIVmac1A11/239env/1A11 and SIVmac239/1A11env/239) of recombinant viruses. Each of the two parental and four recombinant viruses was inoculated intravenously into four rhesus macaques, and all 24 animals were viremic by 4 weeks postinoculation (p.i.). Virus could not be isolated from peripheral blood mononuclear cells (PBMC) of any animals infected with SIVmac1A11 after 6 weeks p.i. but was consistently isolated from all macaques inoculated with SIVmac239 for 92 weeks p.i. Virus isolation was variable from animals infected with recombinant viruses; SIVmac1A11/239gag-env/1A11 and SIVmac239/1A11env/239 were isolated most frequently. Animals inoculated with SIVmac239 had 10 to 100 times more virus-infected PBMC than those infected with recombinant viruses. Three animals infected with SIVmac239 died with simian AIDS (SAIDS) during the 2-year observation period after inoculation, and the fourth SIVmac239-infected animal had clinical signs of SAIDS. Two animals infected with recombinant viruses died with SAIDS; one was infected with SIVmac239/1A11gag-env/239, and the other was infected with SIVmac1A11/239gag-env/1A11. The remaining 18 macaques remained healthy by 2 years p.i., and 13 were aviremic. One year after inoculation, peripheral lymph nodes of some of these healthy, aviremic animals harbored infected cells. All animals seroconverted within the first few weeks of infection, and the magnitude of antibody response to SIV was proportional to the levels and duration of viremia. Virus-suppressive PBMC were detected within 2 to 4 weeks p.i. in all animals but tended to decline as viremia disappeared. There was no association of levels of cell-mediated virus-suppressive activity and either virus load or disease progression. Taken together, these results indicate that differences in more than one region of the viral genome are responsible for the lack of virulence of SIVmac1A11.     

Suboptimal nucleotides in the infectious, pathogenic simian immunodeficiency virus clone SIVmac239

We analyzed virus sequences in two monkeys infected with SIVmac239 and two monkeys infected with SHIVnef that maintained high, persisting viral loads. Sequence changes were observed consistently at four loci in all four animals: a single nucleotide change in the Lys-tRNA primer binding site in the 5' long terminal repeat; two nucleotide changes that resulted in two amino acid changes in the pol gene product; and a single nucleotide change in the region of the simian immunodeficiency virus genome where the rev and env genes overlap, resulting in changes in the predicted amino acid sequences of both gene products. None of these mutations were seen in short-term cultures of CEMx174 cells infected with SIVmac239 or SHIVnef. At all four positions in all four animals, the new sequences represented consensus sequences for primate lentiviruses, whereas the inoculum sequences at these four loci have either never been or rarely been reported outside of SIVmac239. Thus, although cloned SIVmac239 is consistently pathogenic and consistently induces high viral load set points, it is clearly less than optimal at these four nucleotide positions.     

Mechanisms for adaptation of simian immunodeficiency virus to replication in alveolar macrophages

In contrast to the simian immunodeficiency virus SIVmac239, which replicates poorly in rhesus monkey alveolar macrophages, a variant with nine amino acid changes in envelope (SIVmac239/316E) replicates efficiently and to high titer in these same cells. We examined levels of viral DNA, RNA, antigen, and infectious virus to identify the nature of the block to SIVmac239 replication in these cells. Low levels of viral antigen (0.1 to 1.0 ng of p27 per ml) and infectious virus (100 to 1,000 infectious units per ml) were produced in the supernatant 1 to 4 days after SIVmac239 infection, but these levels did not increase subsequently. SIVmac239 DNA was synthesized in these macrophage cultures during the initial 24 h after infection, but the levels did not increase subsequently. Quantitation of the numbers of infectious cells in cultures over time and the results of experiments in which cells were reexposed to SIVmac239 after the initial exposure indicated that only a small proportion of cells were susceptible to SIVmac239 infection in these alveolar macrophage cultures and that the vast majority (>95%) of cells were refractory to SIVmac239 infection. In contrast to the results with SIVmac239, the levels of viral antigen, infectious virus, and viral DNA increased exponentially 2 to 7 days after infection by SIVmac239/316E, reaching levels greater than 100 ng of p27 per ml and 100,000 infectious units per ml. Since SIVmac239/316E has previously been described as a virus capable of infecting cells in a relatively CD4-independent fashion, we examined the levels of CD4 expression on the surface of fresh and cultured alveolar macrophages from rhesus monkeys. The levels of CD4 expression were extremely low, below the limit of detection by flow cytometry, on greater than 99% of the macrophages. CCR5(+) cells were profoundly depleted only from alveolar macrophage cultures infected with SIVmac239/316E. High concentrations of an antibody to CD4 delayed but did not block replication of SIVmac239/316E. The results suggest that the adaptation of SIVmac316 to efficient replication in alveolar macrophages results from its ability to infect these cells in a CD4-independent fashion or in a CD4-dependent fashion even at extremely low levels of surface CD4 expression. Since resident macrophages in brains and lungs of humans also express little or no CD4, our findings predict the presence of human immunodeficiency virus type 1 that is relatively CD4 independent in the lung and brain compartments of infected people.     

Roflumilast enhances cisplatin‐sensitivity and reverses cisplatin‐resistance of ovarian cancer cells via cAMP/PKA/CREB‐FtMt signalling axis

Objective

We previously demonstrated the roflumilast inhibited cell proliferation and increased cell apoptosis in ovarian cancer. In this study, we aimed to investigate the roles of roflumilast in development of cisplatin (DDP)‐sensitive and ‐resistant ovarian cancer.

Methods

OVCAR3 and SKOV3 were selected and the corresponding DDP‐resistant cells were constructed. Cell viability, proliferation, apoptosis, cycle were performed. Expression cAMP, PKA, CREB, phosphorylation of CREB and FtMt were detected. The roles of roflumilast in development of DDP‐sensitive and ‐resistant ovarian cancer were confirmed by xenograft model.

Results

Roflumilast + DDP inhibited cell proliferation, and induced cell apoptosis and G0/G1 arrest in OVCAR3 and SKOV3 cells, roflumilast induced expression of FtMt, the activity of cAMP and PKA and phosphorylation of CREB in ovarian cancer cells and the above‐effect were inhibited by H89. Downregulation of CREB inhibited the roflumilast‐increased DDP sensitivity of ovarian cancer cells, and the roflumilast‐induced FtMt expression and phosphorylation of CREB. Also, roflumilast reversed cisplatin‐resistance, and induced expression of FtMt and activation of cAMP/PKA/CREB in DDP‐resistant ovarian cancer cells. Similarly, treated with H89 or downregulation of CREB inhibited the changes induced by roflumilast. In vivo, roflumilast inhibited the development of SKOV3 or SKOV3‐DDP‐R xenograft models.

Conclusions

Roflumilast enhanced DDP sensitivity and reversed the DDP resistance of ovarian cancer cells via activation of cAMP/PKA/CREB pathway and upregulation of the downstream FtMt expression, which has great promise in clinical treatment.

     

Transmodulation of epidermal growth factor receptor function by cyclic AMP-dependent protein kinase.

Binding of epidermal growth factor (EGF) to its receptor (EGFR) augments the tyrosine kinase activity of the receptor and autophosphorylation. Exposure of some tissues and cells to EGF also stimulates adenylyl cyclase activity and results in an increase in cyclic AMP (cAMP) levels. Because cAMP activates the cAMP-dependent protein kinase A (PKA), we investigated the effect of PKA on the EGFR. The purified catalytic subunit of PKA (PKAc) stoichiometrically phosphorylated the purified full-length wild type (WT) and kinase negative (K721M) forms of the EGFR. PKAc phosphorylated both WT-EGFR as well as a mutant truncated form of EGFR (Delta1022-1186) exclusively on serine residues. Moreover, PKAc also phosphorylated the cytosolic domain of the EGFR (EGFRKD). Phosphorylation of the purified WT as well as EGFRDelta1022-1186 and EGFRKD was accompanied by decreased autophosphorylation and diminished tyrosine kinase activity. Pretreatment of REF-52 cells with the nonhydrolyzable cAMP analog, 8-(4-chlorophenylthio)-cAMP, decreased EGF-induced tyrosine phosphorylation of cellular proteins as well as activation of the WT-EGFR. Similar effects were also observed in B82L cells transfected to express the Delta1022-1186 form of EGFR. Furthermore, activation of PKAc in intact cells resulted in serine phosphorylation of the EGFR. The decreased phosphorylation of cellular proteins and diminished activation of the EGFR in cells treated with the cAMP analog was not the result of altered binding of EGF to its receptors or changes in receptor internalization. Therefore, we conclude that PKA phosphorylates the EGFR on Ser residues and decreases its tyrosine kinase activity and signal transduction both in vitro and in vivo.