Mesenchymal Stromal Cells Prevent Allostimulation In Vivo and Control Checkpoints of Th1 Priming: Migration of Human DC to Lymph Nodes and NK Cell Activation.

Although the immunomodulatory potency of mesenchymal stromal cells (MSC) is well established, the mechanisms behind are still not clear. The crosstalk between myeloid dendritic cells (mDC) and natural killer (NK) cells and especially NK cell-derived interferon-gamma (IFN-γ) play a pivotal role in the development of type 1 helper (Th1) cell immune responses. While many studies explored the isolated impact of MSC on either in vitro generated DC, NK, or T cells, there are only few data available on the complex interplay between these cells. Here, we investigated the impact of MSC on the functionality of human mDC and the consequences for NK cell and Th1 priming in vitro and in vivo. In critical limb ischemia patients, who have been treated with allogeneic placenta-derived mesenchymal-like stromal cells (PLX-PAD), no in vivo priming of Th1 responses toward the major histocompatibility complex (MHC) mismatches could be detected. Further in vitro studies revealed that mDC reprogramming could play a central role for these effects. Following crosstalk with MSC, activated mDC acquired a tolerogenic phenotype characterized by reduced migration toward CCR7 ligand and impaired ability to stimulate NK cell-derived IFN-γ production. These effects, which were strongly related to an altered interleukin (IL)-12/IL-10 production by mDC, were accompanied by an effective prevention of Th1 priming in vivo. Our findings provide novel evidence for the regulation of Th1 priming by MSC via modulation of mDC and NK cell crosstalk and show that off-the-shelf produced MHC-mismatched PLX-PAD can be used in patients without any sign of immunogenicity.

Meeting report of the first conference of the International Placenta Stem Cell Society (IPLASS).

The International Placenta Stem Cell Society (IPLASS) was founded in June 2010. Its goal is to serve as a network for advancing research and clinical applications of stem/progenitor cells isolated from human term placental tissues, including the amnio-chorionic fetal membranes and Wharton's jelly. The commitment of the Society to champion placenta as a stem cell source was realized with the inaugural meeting of IPLASS held in Brescia, Italy, in October 2010. Officially designated as an EMBO-endorsed scientific activity, international experts in the field gathered for a 3-day meeting, which commenced with "Meet with the experts" sessions, IPLASS member and board meetings, and welcome remarks by Dr. Ornella Parolini, President of IPLASS. The evening's highlight was a keynote plenary lecture by Dr. Diana Bianchi. The subsequent scientific program consisted of morning and afternoon oral and poster presentations, followed by social events. Both provided many opportunities for intellectual exchange among the 120 multi-national participants. This allowed a methodical and deliberate evaluation of the status of placental cells in research in regenerative and reparative medicine. The meeting concluded with Dr. Parolini summarizing the meeting's highlights. This further prepared the fertile ground on which to build the promising potential of placental cell research. The second IPLASS meeting will take place in September 2012 in Vienna, Austria. This meeting report summarizes the thought-provoking lectures delivered at the first meeting of IPLASS.

Treatment development for systemic Tetrahymena sp. infection in guppies, Poecilia reticulata Peters.

Antibacterial and antiparasitic agents and a cysteine protease inhibitor (E-64) were tested against Tetrahymena infection, a serious problem in guppy production worldwide. Chemicals were tested in vitro by a colorimetric assay for Tetrahymena survival. The most effective were niclosamide, albendazole and chloroquine, with 23%, 35% and 60% survival, respectively, following 2-h exposure to 100 ppm. Longer incubation periods resulted in greater reductions in survival. Niclosamide was further studied in vivo at different dosages, administered orally to Tetrahymena-infected guppies. Mortality rates were significantly lower in all treatment groups; in trial I, 30% and 33% mortality in 5 and 40 mg kg(-1) niclosamide-fed fish vs. 59% mortality in controls; in trial II, 35%, 13% and 10% in 50, 100 and 200 mg kg(-1) niclosamide-fed fish vs. 64% in controls. The effect of the cysteine protease inhibitor E64 was tested in tissue culture, by measuring histolytic activity of the parasite (Tet-NI) on a guppy-fin cell line, based on cell depletion. Tet-NI feeding activity was significantly reduced following pretreatment with E-64 relative to non-treated Tet-NI. E-64-pretreated Tet-NI was injected i.p. into guppies: recorded mortality rates were significantly lower (35%) than that in non-treated Tet-NI (60%), suggesting inhibition of the parasite's cysteine protease as a possible therapeutic approach.

Cysteine proteases and acid phosphatases contribute to Tetrahymena spp. pathogenicity in guppies, Poecilia reticulata.

Systemic tetrahymenosis caused by the protozoan parasite Tetrahymena spp. is a serious problem in guppy (Poecilia reticulata) farms worldwide. There is no therapeutic solution for the systemic form of this disease. Guppies severely infected with Tetrahymena spp. were imported by a commercial ornamental fish farm and brought to our laboratory. Tetrahymena sp. (Tet-NI) was isolated and in vitro cultured. Isolates maintained in culture for different time periods (as reflected by different numbers of passages in culture) were analyzed-Tet-NI 1, 4, 5 and 6, with Tet-NI 1 being cultured for the longest period (about 15 months, 54 passages) and Tet-NI 6 for the shortest (2.5 months, 10 passages). Controlled internal infection was successfully achieved by IP injection with most isolates, except for Tet-NI 1 which produced no infection. The isolate Tet-NI 6 induced the highest infection rates in internal organs (80% vs. 50% and 64% for Tet-NI 4 and 5, respectively) and mortality rates (67% vs. 20% and 27% for Tet-NI 4 and 5, respectively, and 6.7% for Tet-NI 1). The correlation between pathogenicity and Tetrahymena enzymatic activity was studied. Electrophoretic analyses revealed at least two bands of gelanolytic activity in Tet-NI 4 and 5, three bands in Tet-NI 6, and no activity in Tet-NI 1. Total inhibition of gelanolytic activity was observed after pretreatment of Tet-NI 6 with E-64, a highly selective cysteine protease inhibitor. Using hemoglobin as a substrate, Tet-NI 6 had two bands of proteolytic activity and no bands were observed in Tet-NI 1. A correlation was observed between pathogenicity and acid phosphatase activities (analyzed by commercial fluorescence kit) for Tet-NI 1 and Tet-NI 6.

Protective immunization against Tetrahymena sp. infection in guppies (Poecilia reticulata).

Systemic tetrahymenosis constitutes a serious problem in guppy (Poecilia reticulata) production worldwide and no therapeutic solution is available for this disease. Three immunization trials were conducted, testing the effectiveness of different Tetrahymena preparations applied by intraperitoneal injection (IP) with or without Freund's complete adjuvant (FCA) and with or without booster dose. In trial 1, immunization with the pathogenic Tet-NI 6 lysate and live attenuated Tet-NI 1 did not provide significant protection from infection, although infection rates were significantly lower in the Tet-NI 6-immunized group than in controls. In trial 2, mortality in Tet-NI 6 + FCA-immunized fish was 10%, significantly lower than in all other treatment groups, including Tet-NI 6 lysate, live attenuated Tet-NI 1 and controls (77, 67 and 73%, respectively). In trial 3, the lowest mortality rates were obtained in the Tet-NI 6 + FCA + booster-immunized group (15%). These levels were lower but not significantly different from the non-boostered Tet-NI 6-immunized group (28%) and the groups immunized with Tet-NI 1, with and without booster (32 and 34%, respectively). Mortality in these four groups was significantly lower than in controls, including adjuvant- and PBS-injected groups (72 and 81%, respectively). Body homogenates of immunized fish immobilized Tetrahymena in-vitro, as compared to no or very little immobilization in controls. Lysozyme levels in the Tet-NI 6 + FCA + booster group were significantly higher than in all other treatments in trial 2 and controls in trial 3. There was no significant difference in anti-protease activity among the differently immunized fish. We conclude that immunization with Tetrahymena lysates in FCA confers a high degree of protection from infection, suggesting this preparation as a basis for vaccine development.

Characterization of swim bladder non-inflation (SBN) in angelfish, Pterophyllum scalare (Schultz), and the effect of exposure to methylene blue.

Failure to inflate the swim bladder is regarded a major obstacle in the rearing of many fish species. We present a study of swim bladder non-inflation (SBN) in angelfish, Pterophyllum scalare. A normal developing primordial swim bladder was first discernable at the end of the first day post-hatch (p.h.) as a cluster of epithelial cells with a central lumen, surrounded by presumably mesenchymal cells. Initial inflation occurred on the fourth day p.h. Prior to inflation the swim bladder epithelium consisted of an outer squamous and inner columnar layer. Cells of the inner layer were filled at their basal region with an amorphous material, which disappeared upon inflation. A pneumatic duct was absent, and larvae presented no need to reach the water surface for inflation, suggesting that angelfish are pure physoclists. A model for the role of the amorphous material in normal initial inflation is proposed. Abnormal swim bladders were apparent from the fourth day p.h., and methylene blue (MB) at a concentration of 5 ppm significantly increased the prevalence of SBN. Histologically, abnormal swim bladders in larvae hatched in 5 ppm MB could not be distinguished from those in fish raised under routine conditions (0.5 ppm MB). We suggest that MB may have a teratogenic effect in angelfish.

Taxol-induced apoptosis in human SKOV3 ovarian and MCF7 breast carcinoma cells is caspase-3 and caspase-9 independent.

Taxol is used in chemotherapy regimens against breast and ovarian cancer. Treatment of tumor model cell lines with taxol induces apoptosis, but exact mechanism is not sufficiently understood. Our results demonstrate that in response to taxol, various cell types differentially utilize distinct apoptotic pathways. Using MCF7 breast carcinoma cells transfected with caspase-3 gene, we showed that taxol-induced apoptosis occurred in the absence of caspase-3 and caspase-9 activation. Similar results were obtained with ovarian SKOV3 carcinoma cells, expressing high level of endogenous caspase-3. In contrast, staurosporine-induced apoptosis in these cells was accompanied by proteolytic cleavage of pro-caspase-3 and induction of caspase-3 enzymatic activity. The effect of taxol appears to be cell type-specific, since taxol-induced apoptosis in leukemia U937 cells involved caspase-3 activation step. We conclude that a unique caspase-3 and caspase-9 independent pathway is elicited by taxol to induce apoptosis in human ovarian and breast cancinoma cells.

Gamma-radiation-induced growth arrest and apoptosis in p53-null lymphoma cells is accompanied by modest transcriptional changes in many genes.

Damage to DNA produces cell cycle arrest, apoptosis, or both. The response in cells with p53 tumor suppressor function involves transcriptional changes, but whether that holds for cells lacking active p53, as in most tumors, is not known. Better characterization of the DNA damage response in tumors lacking p53 function is relevant to cytotoxic therapy. We have explored whether gamma-irradiated p53-null mouse T lymphoma cells undergo marked changes in transcription. Their arrest in G2/M prior to apoptosis required transcription. Transcripts whose abundance altered on irradiation were sought by subtractive hybridization, and 1010 candidate clones from two oppositely enriched cDNA populations were sequenced. Hybridization revealed small (<3-fold) increases or decreases in the transcripts of more than 15 genes, including some implicated in cell cycle control (e.g., BTG, Bap1) or apoptosis (e.g., STAT1, calpain), but no marked changes like those associated with other forms of T-cell death. Moreover, the expression of some critical apoptosis regulators, such as Bcl-2 family members, did not change. Hence, the G2/M arrest and apoptosis in the irradiated p53-null lymphoma appears to involve modest expression changes for many genes, but post-transcriptional alterations may be more critical.

Position effect of human telomeric repeats on replication timing.

Telomeres are distinct structures, composed of short, repeated sequences, at the ends of all eukaryotic chromosomes. Telomeres have been shown in yeast to induce late replication in S phase and to silence transcription of neighboring genes. To examine the possibility of similar effects in human chromosomes, we studied cells from a subject with a microdeletion of 130 kb at the end of one copy of chromosome arm 22q, repaired by the addition of telomere repeats. Using fluorescence in situ hybridization of S phase nuclei, a distinct difference was found in the replication timing of the breakpoint region between the intact and truncated copies of chromosome 22. This difference was evident as a shift from middle to late replication time of the breakpoint region adjacent to the repaired telomere. This finding suggests that the human telomere sequence influences activation of adjacent replication origin(s). The difference in replication timing between the two chromosomes was not associated with differences in sensitivity to digestion by DNase I or with methylation of regions immediately adjacent to the breakpoint. Furthermore, both alleles of arylsulfatase A, a gene located at a distance of approximately 54 kb from the breakpoint, were expressed. We conclude that as in yeast, the proximity of telomeric DNA may induce a positional effect that delays the replication of adjacent chromosomal regions in humans.

Antitumor and immunotherapeutic effects of activated invasive T lymphoma cells that display short-term interleukin 1alpha expression.

Expression of cytokines in malignant cells represents a novel approach for therapeutic treatment of tumors. Previously, we demonstrated the immunostimulatory effectiveness of interleukin 1alpha (IL-1alpha) gene transfer in experimental fibrosarcoma tumors. Here, we report the antitumor and immunotherapeutic effects of short-term expression of IL-1alpha by malignant T lymphoma cells. Activation in culture of T lymphoma cells with lipopolysaccharide-stimulated macrophages induces the expression of IL-1alpha. The short-term expression of IL-1alpha persists in the malignant T cells for a few days (approximately 3-6 days) after termination of the in vitro activation procedure and, thus, has the potential to stimulate antitumor immune responses in vivo. As an experimental tumor model, we used the RO1 invasive T lymphoma cell line. Upon i.v. inoculation, these cells invade the vertebral column and compress the spinal cord, resulting in hind leg paralysis and death of the mice. Activated RO1 cells, induced to express IL-1alpha in a short-term manner, manifested reduced tumorigenicity: approximately 75% of the mice injected with activated RO1 cells remained tumor free. IL-1 was shown to be essential for the eradication of activated T lymphoma cells because injection of activated RO1 cells together with IL-1-specific inhibitors, i.e., the IL-1 receptor antagonist or the M 20 IL-1 inhibitor, reversed reduced tumorigenicity patterns and led to progressive tumor growth and death of the mice. Furthermore, activated RO1 cells could serve as a treatment by intervening in the growth of violent RO1 cells after tumor take. Thus, when activated RO1 cells were injected 6 or 9 days after the inoculation of violent cells, mortality was significantly reduced. IL-1alpha, in its unique membrane-associated form, in addition to its cytosolic and secreted forms, may represent a focused adjuvant for potentiating antitumor immune responses at low levels of expression, below those that are toxic to the host. Further assessment of the immunotherapeutic potential of short-term expression of IL-1alpha in activated tumor cells may allow its improved application in the treatment of malignancies.

Interference with gene expression induces rapid apoptosis in p53-null T lymphoma cells.

Two p53-null T lymphoma cell lines proved to be highly sensitive to inhibition of gene expression. With either actinomycin D or cycloheximide, apoptosis commenced within 2 h, as indicated by loss of membrane integrity, degradation of certain proteins (including the phosphatase calcineurin) and DNA fragmentation. These effects were ablated by co-expression of Bcl-2 or co-incubation with the caspase inhibitor Z-VAD-fmk. These results suggest that the apoptotic machinery is in place in these cells but held in check by an unknown labile protein, which probably acts upstream of Bcl-2. Although cycloheximide can activate the JNK or p38 MAP kinases in some cells, neither was implicated here. However, disruption of phosphoinositide 3-kinase signaling may be involved, because the cells were also sensitive to wortmannin. The high sensitivity of the p53-null lymphoma cells to inhibitors of gene expression suggests that such inhibitors might prove useful in the cytotoxic therapy of certain tumors.

The ftsZ gene as a tool for detection of Mycoplasma fermentans.

Mycoplasma fermentans was reported as a common contaminant of cell cultures, and was shown to either induce or suppress several immunological functions. A strain of M. fermentans was recently isolated from a mouse T-lymphoma cell line, which differs from other M. fermentans strains by its growth characteristics and was designated (in the authors' records) as strain 609. Using the differential display technique (DD), a differentially expressed gene that was identified as the M. fermentans 609 ftsZ gene was isolated. Comparison of the nucleotide sequence of the M. fermentans 609 ftsZ gene to other ftsZ genes showed a 98% homology with Mycoplasma fermentans strain K7 and approximately 50% homology with Mycoplasma pulmonis and Mycoplasma genitalium. Comparison of the putative amino acid sequences of the FtsZ proteins showed similar homology. A polymerase chain reaction (PCR) assay to detect the presence of this ftsZ gene was established; it is a fast and convenient assay to detect infection of cells by the M. fermentans species. This work demonstrates that: (i) DD can be used as a useful technique to identify and isolate mycoplasmal genes from infected cells; and (ii) the ftsZ gene can be a useful marker to distinguish between different species of mycoplasma.

Identification of genes induced by interleukin-3 and erythropoietin via the Jak-Stat5 pathway using enhanced differential display-reverse southern.

Cytokines mediate their effects on growth and maturation of hematopoietic cells by binding to their cognate receptors and activating target genes. Interleukin-3 (IL-3) and erythropoietin (Epo) induce signal transduction via the Jak-Stat pathway. We report here on the identification of several known and novel genes induced by IL-3 and Epo, using a modified version of the PCR-based technique, enhanced differential display (EDD). We modified the technique to facilitate the screening and verification of the differential expression of the genes by using reverse Southern blotting (RS) and PCR-Southern blotting, and we called it EDD-RS. From the initial 110 genetags that were identified as differential expressed genes, 14 contained more than one gene. Among the differentially expressed genes, 24 are known genes and 39 are novel genes. Several of the known genes, such as IRF-1 and P21waf, were previously observed by others to be induced by IL-3 and Epo, but their dependence on Stat5 activation in cytokine-dependent cells was unknown. Other known genes, such as crp and Mssp2/1, were not described previously as target genes for cytokine induction. The results demonstrate that EDD-RS is an efficient method to identify cytokine-induced genes and can be productive in delineating the signal required for their induction.

The incidence and possible relevance of Y-linked microdeletions in babies born after intracytoplasmic sperm injection and their infertile fathers.

Microdeletions linked to deletion intervals 5 and 6 of the Y chromosome have been associated with male factor infertility. Members from at least two gene families lie in the region containing azoospermia factor (AZF), namely YRRM and DAZ. With the advent of intracytoplasmic sperm injection (ICSI), it is possible for men with severe male factor infertility to produce a child. The genetic consequences of such a procedure have been questioned. This report describes the first study of a population (32 couples) of infertile fathers and their sons born after ICSI. The objectives were firstly to determine the incidence and map location of Y chromosome microdeletions and to compare the frequencies with other population studies involving severe male factor infertility, and secondly to formulate a working hypothesis concerning developmental aetiology of Y chromosome microdeletions. The incidence of microdeletions in the ICSI population was shown to be 9.4% (within the range 9-18% reported for populations of severe male factor infertility patients). Microdeletions in two out of three affected father/son pairs mapped in the region between AZFb and AZFc and the third involved a large microdeletion in AZFb and AZFc. Of three affected father/son pairs, microdeletions were detected in the blood of one infertile propositus father and three babies. Assuming that the gonomes of the ICSI-derived babies are direct reflections of those of their fathers germ lines, it is possible that two of three infertile fathers were mosaic for intact Y and microdeleted Y chromosomes. In such cases, the developmental aetiology of the microdeletion may be due to a de-novo microdeletion arising as a post-zygotic mitotic error in the infertile propositus father, thus producing a mosaic individual who may or may not transmit the deletion to his ICSI-derived sons depending on the extent of primordial germ cell mosaicism. In one of three affected fathers, the microdeletion detected in his blood was also detected in his ICSI-derived son. In this case the de-novo event giving rise to the microdeletion may have occurred due to a post- (or pre-) meiotic error in the germ line of this father's normally fertile father (i.e. the ICSI-derived baby's grandfather).

Tyrosine phosphorylation of measles virus P-phosphoprotein in persistently infected neuroblastoma cells.

Replication and encapsidation of measles virus (MV) requires the interaction between the nuclear protein (N) and the phosphoprotein (P). It is known that both proteins are phosphorylated on serine and threonine residues. Recently we have shown that N is phosphorylated on tyrosine in persistently-infected mouse neuroblastoma cells (NS20Y/MS). Here, we show that P in NS20Y/MS is also phosphorylated on tyrosine. To investigate whether cellular tyrosine kinases can bind and phosphorylate P, a solid phase kinase assay was employed. We show that bacterially-expressed MV P fragments, were phosphorylated on tyrosine by purified mouse c-Src protein-tyrosine kinase and when mixed with uninfected neuroblastoma cell (NS20Y) extracts, these P fragments were phosphorylated on tyrosine in addition to serine and threonine. These results imply that MV P is a substrate for tyrosine phosphorylation by cellular tyrosine kinase(s).

Structural and functional analysis of the promoter of the murine V gamma 1.1 T cell receptor gene.

The expression of the germ-line gene V gamma 1.1-C gamma 4 of the T cell receptor (TcR) gamma chain depends on interleukin (IL)-3 induction in hematopoietic cells, while in T cells, the rearranged gene is expressed constitutively. To understand the mechanism that controls TcR gamma gene expression, we cloned and characterized the structure and function of the V gamma 1.1-C gamma 4 TcR promoter. IL-3-dependent cell lines and T cell lines utilized the same transcriptional start sites. In chloramphenicol acetyltransferase (CAT) assays, the minimal 70-bp promoter confers strong transcriptional activity which is 50-60% of the Moloney long terminal repeat promoter activity. The 500-bp promoter region linked to the CAT gene exhibits IL-3 dependency similar to the endogenous TcR gamma gene. The immediate 3' and 5' flanking sequences inhibit the promoter activity two- to fourfold. The promoter lacks an obvious TATA box or CAAT box sequences, but contains a GC box in the untranslated region 3' to the promoter. The GC box is the core sequence of the element which binds Sp1-like proteins. Cloning of this Sp1 binding element in front of the thymidine kinase (TK) promoter and mutations generated in this site demonstrate its function as a silencer. Ultraviolet cross-linking analysis with the Sp1 binding site from the TcR gamma promoter revealed binding of a 90-100-kDa protein in a T cell line (EL-4) and 40-50 and 90-100-kDa proteins in FDC-P1 cells. The possible function of the Sp1-like protein in silencing the minimal promoter activity is discussed.

Tyrosine phosphorylation of measles virus nucleocapsid protein in persistently infected neuroblastoma cells.

Subacute sclerosing panencephalitis is a slowly progressing fatal human disease of the central nervous system which is a delayed sequel of measles virus (MV) infection. A typical pathological feature of this disease is the presence of viral ribonucleocapsid structures in the form of inclusion bodies and the absence of infectious virus or budding viral particles. The mechanisms governing the establishment and maintenance of a persistent MV infection in brain cells are still largely unknown. To understand the mechanisms underlying MV persistence in neuronal cells, a tissue culture model was studied. Clone NS20Y/MS of the murine neuroblastoma C1300 persistently infected with the wild-type Edmonston strain of MV secretes relatively high levels of alpha/beta interferon (IFN). As shown previously, treatment of the persistently infected cultures with anti-IFN serum converted the persistent state into a productive infection indicated by the appearance of multinucleated giant cells. In this study, we have investigated whether alpha/beta IFN produced by NS20Y/MS cells activates cellular protein tyrosine kinases which will induce tyrosine phosphorylating activity specific to virus-infected cells. We present data to show augmented protein tyrosine kinase activity in the persistently infected cells. We demonstrate that the MV N protein is phosphorylated on tyrosine in addition to serine and threonine in the persistent state but not in NS20Y cells acutely infected with MV.

Oligoclonality of Moloney leukemias.

Induction of leukemia by non-transforming retroviruses results in the appearance of various hematopoietic tumors. It is believed that these tumors are monoclonal. In this work, the clonal nature of Moloney leukemia virus (MoLV)-induced tumors was studied. Two genetic parameters were used in order to identify leukemic clones: the pattern of the proviral integration sites and the rearrangement of the T-cell receptor complex (TCR). In more than 60% of the mice, different leukemic clones populated tumors developed in different organs of the same animal. Genotypic analysis of cell lines derived from a leukemic organ revealed that the tumor is composed of more than one clone. Phenotypic analysis of subclones which were derived from a monoclonal cell line showed variability in the expression of the Thy 1.2 and MHC antigens. The results indicate that MoLV-induced tumors are of oligoclonal nature. Each leukemic organ contains a mixture of leukemic clones, of which one is dominant.

Alteration of a cyclic AMP-dependent protein kinase phosphorylation site in the c-Fos protein augments its transforming potential.

We have studied the phosphorylation of the nuclear oncoprotein Fos by cyclic AMP-dependent protein kinase (PKA). We demonstrate that the human c-Fos protein, phosphorylated either in vitro with purified PKA or in vivo in JEG3 cells following treatment with forskolin, has similar phosphotryptic peptide maps. Serine 362, which constitutes part of a canonical PKA phosphorylation site (RKGSSS), is phosphorylated both in vivo and in vitro. A mutant of Fos protein in which serine residues 362 to 364 have been altered to alanine residues is not efficiently phosphorylated in vitro. Furthermore, Fos protein in which serines 362 to 364 have been altered to alanine shows increased transforming potential. We propose that phosphorylation of Fos by PKA is an important regulatory step in controlling its activity in normal cell growth and differentiation.