Xeno-free trans-differentiation of adipose tissue-derived mesenchymal stem cells into glial and neuronal cells.

Mesenchymal stem cells (MSCs) are undifferentiated cells that have the ability of self-renewal and trans-differentiation into other cell types. They hold out hope for finding a cure for many diseases. Nevertheless, there are still some obstacles that limit their clinical transplantation. One of these obstacles are the xenogeneic substances added in either proliferation or differentiation media with subsequent immunogenic and infectious transmission problems. In this study, we aimed to replace fetal bovine serum (FBS), the main nutrient source for MSC proliferation with xeno-free blood derivatives. We tested the effect of human activated pure platelet-rich plasma (P-PRP) and advanced platelet-rich fibrin (A-PRF) on the proliferation of human adipose derived-MSCs (AD-MSCs) at different concentrations. For the induction of MSC neural differentiation, we used human cerebrospinal fluid (CSF) at different concentrations in combination with P-PRP to effect xeno-free/species-specific neuronal/glial differentiation and we found that media with 10% CSF and 10% PRP promoted glial differentiation, while media with only 10% PRP induced a neuron-like phenotype.

Potential of Stem Cell-Based Therapy for Ischemic Stroke.

Ischemic stroke is one of the major health problems worldwide. The only FDA approved anti-thrombotic drug for acute ischemic stroke is the tissue plasminogen activator. Several studies have been devoted to assessing the therapeutic potential of different types of stem cells such as neural stem cells (NSCs), mesenchymal stem cells, embryonic stem cells, and human induced pluripotent stem cell-derived NSCs as treatments for ischemic stroke. The results of these studies are intriguing but many of them have presented conflicting results. Additionally, the mechanism(s) by which engrafted stem/progenitor cells exert their actions are to a large extent unknown. In this review, we will provide a synopsis of different preclinical and clinical studies related to the use of stem cell-based stroke therapy, and explore possible beneficial/detrimental outcomes associated with the use of different types of stem cells. Due to limited/short time window implemented in most of the recorded clinical trials about the use of stem cells as potential therapeutic intervention for stroke, further clinical trials evaluating the efficacy of the intervention in a longer time window after cellular engraftments are still needed.

Genetically unmatched human iPSC and ESC exhibit equivalent gene expression and neuronal differentiation potential.

The potential uniformity between differentiation and therapeutic potential of human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) remains debatable. We studied the gene expression profiles, pathways analysis and the ability to differentiated into neural progenitor cells (NPCs) and motor neurons (MNs) of genetically unmatched integration-free hiPSC versus hESC to highlight possible differences/similarities between them at the molecular level. We also provided the functional information of the neurons derived from the different hESCs and hiPSCs lines using the Neural Muscular Junction (NMJ) Assay. The hiPSC line was generated by transfecting human epidermal fibroblasts (HEF) with episomal DNAs expressing Oct4, Sox2, Klf4, Nanog, L-Myc and shRNA against p53. For the hESCs line, we used the NIH-approved H9 cell line. Using unsupervised clustering both hESCs and hiPSCs were clustered together implying homogeneous genetic states. The genetic profiles of hiPSCs and hESCs were clearly similar but not identical. Collectively, our data indicate close molecular similarities between genetically unmatched hESCs and hiPS in term of gene expression, and signaling pathways. Moreover, both cell types exhibited similar cholinergic motor neurons differentiation potential with marked ability of the differentiated hESCs and hiPSCs-derived MNs to induce contraction of myotubes after 4 days of co-culture.

Therapeutic potential of human olfactory bulb neural stem cells for spinal cord injury in rats.

STUDY DESIGN: Adult human olfactory bulb neural stem cells (OBNSCs) were isolated from human patients undergoing craniotomy for tumor resection. They were genetically engineered to overexpresses green fluorescent protein (GFP) to help trace them following engraftment. Spinal cord injury (SCI) was induced in rats using standard laminectomy protocol, and GFP-OBNSC were engrafted into rat model of SCI at day 7 post injury. Three rat groups were used: (i) Control group, (ii) Sham group (injected with cerebrospinal fluid) and treated group (engrafted with OBNSCs). Tissues from different groups were collected weekly up to 2 months. The collected tissues were fixed in 4% paraformaldehyde, processed for paraffin sectioning, immunohistochemically stained for different neuronal and glial markers and examined with bright-field fluorescent microscopy. Restoration of sensory motor functions we assessed on a weekly bases using the BBB score. OBJECTIVES: To assess the therapeutic potential of OBNSCs-GFP and their ability to survive, proliferate, differentiate and to restore lost sensory motor functions following their engraftment in spinal cord injury (SCI). METHODS: GFP-OBNSC were engrafted into a rat model of SCI at day 7 post injury and were followed-up to 8 weeks using behavioral and histochemical methods. RESULTS: All transplanted animals exhibited successful engraftment. The survival rate was about 30% of initially transplanted cells. Twenty-seven percent of the engrafted cells differentiated along the NG2 and O4-positive oligodendrocyte lineage, 16% into MAP2 and ß-tubulin-positive neurons, and 56% into GFAP-positive astrocytes. CONCLUSION: GFP-OBNSCs had survived for >8 weeks after engraftment and were differentiated into neurons, astrocytes and oligodendrocytes, The engrafted cells were distributed throughout gray and white matter of the cord with no evidence of abnormal morphology or any mass formation indicative of tumorigenesis. However, the engrafted cells failed to restore lost sensory and motor functions as evident from behavioral analysis using the BBB score test.

Role of Bim in apoptosis induced in H460 lung tumor cells by the spindle poison Combretastatin-A4.

The BH3-only Bcl-2 subfamily member Bim is a well known apoptosis promoting protein. However, the mechanisms upstream of mitochondrion membrane permeability by which Bim is involved in apoptosis have been poorly investigated, particularly in response to agents capable of interfering with the cytoskeleton architecture and arresting cells in mitosis. Based on the observation that Bim is sequestered on the microtubule-array by interaction with the light chain of dynein, we have investigated upon depolymerisation, whether Bim could be involved in the commitment of apoptosis. With this purpose H460 Non Small Lung Cancer Cells (NSLC) were treated with the microtubule damaging agent combretastatin-A4 (CA-4) (7.5 nM; 8-48 h), and various parameters were investigated. Upon treatment, cells arrested in mitosis and died through a caspase-3-dependent mitotic catastrophe. Transient knock down of Bim drastically reduced apoptosis, indicating that this protein was involved in cell death as induced by microtubules disorganisation. In response to increasing conditions of microtubules depolymerisation, we found that the protein level of Bim was strongly upregulated in a time-dependent manner at transcriptional level. Furthermore, Bim was released from microtubule-associated components. Bim was translocated to mitochondria, even in a condition of protein synthesis inhibition, where it showed a markedly increased interaction with Bcl-2. In turn, the fraction of Bax bound to Bcl-2 decreases in response to treatment, thereby indicating that Bim possibly promotes Bax release from the pro-survival protein Bcl-2. Overall, we demonstrated that Bim is required for the CA-4-induced cell death in the H460 lung cancer cell line via activation of the mitochondrial signalling pathway. Defining the contribution of Bim to the mechanism of apoptosis may offer some different clues in view of developing new strategies for chemotherapy with CA-4, underlining the relevance of the cytoskeleton integrity in the apoptotic response.

Monoclonal antibodies as a tool for structure-function studies of the MDR1-P-glycoprotein.

P-glycoprotein is considered one of the most important member of the rapidly growing superfamily of integral proteins known as the ATP-binding cassette (ABC) which in human also include several other multidrug resistance membrane proteins (i.e., MRP), the product of the cystic fibrosis gene, the TAP-1/TAP2 peptide transporters encoded by the major histocompatibility complex genes and the gene encoding for breast cancer resistance protein (BCRP) also known as MXR1 (mitoxantrone resistance protein). Many monoclonal antibodies (MAbs) reacting with distinct P-glycoprotein domains have been isolated and used to study the molecular organization and cellular functions of this ABC protein. MAbs have been used for multidrug resistance (mdr) gene cloning, delineation of the secondary and tertiary structure of P-glycoprotein and molecular analysis of the mechanisms involved in substrate recognition and transport. The immunodetection of the distinct products of the mdr gene family in normal and malignant cells and tissues has greatly contributed to the understanding of the physiological role of P-glycoprotein and its possible involvement in the refractory of tumors to chemotherapy. The present article deals with the immunological methods used for the structure-function studies of the P-glycoprotein. After introducing the basic structural features of this ABC transporter, the antibody based-approach is discussed with aiming to furnishing methodological perspectives for further investigations of the physiological role of P-glycoprotein and the multidrug resistance phenomenon.

Human T cell leukemia virus type 1 Tax associates with a molecular chaperone complex containing hTid-1 and Hsp70.

Tax, an oncogenic viral protein encoded by human T cell leukemia virus type 1 (HTLV-1), induces cellular transformation of T lymphocytes by modulating a variety of cellular gene expressions [1]. Identifying cellular partners that interact with Tax constitutes the first step toward elucidating the molecular basis of Tax-induced transformation. Here, we report a novel Tax-interacting protein, hTid-1. hTid-1, a human homolog of the Drosophila tumor suppressor protein Tid56, was initially characterized based on its interaction with the HPV-16 E7 oncoprotein [2]. hTid-1 and Tid56 are members of the DnaJ family [2,3], which contains a highly conserved signature J domain that regulates the activities of heat shock protein 70 (Hsp70) by serving as cochaperone [4-6]. In this context, the molecular chaperone complex is involved in cellular signaling pathways linked to apoptosis, protein folding, and membrane translocation and in modulation of the activities of tumor suppressor proteins, including retinoblastoma, p53, and WT1[7-12]. We find that expression of hTid-1 inhibits the transformation phenotype of two human lung adenocarcinoma cell lines. We show that Tax interacts with hTid-1 via a central cysteine-rich domain of hTid-1 while a signature J domain of hTid-1 mediates its binding to Hsp70 in HEK cells. Importantly, Tax associates with the molecular chaperone complex containing both hTid-1 and Hsp70 and alters the cellular localization of hTid-1 and Hsp70. In the absence of Tax, expression of the hTid-1/Hsp70 molecular complex is targeted to perinuclear mitochondrial clusters. In the presence of Tax, hTid-1 and its associated Hsp70 are sequestered within a cytoplasmic "hot spot" structure, a subcellular distribution that is characteristic of Tax in HEK cells.

Five human genes encoding F-box proteins: chromosome mapping and analysis in human tumors.

Members of the F-box protein (Fbp) family are characterized by an approximately 40 amino acid F-box motif. SCF complexes (formed by Skp1, cullin, and one of many Fbps) act as protein-ubiquitin ligases that control the G(1)/S transition of the eukaryotic cell cycle. The substrate specificity of SCF complexes is determined by the presence of different Fbp subunits that recruit specific substrates for ubiquitination. Unchecked degradation of cellular regulatory proteins has been observed in certain tumors and it is possible that deregulated ubiquitin ligases play a role in the altered degradation of cell cycle regulators. We have recently identified a family of human Fbps. As a first step aimed at determining if FBP genes could be involved in human neoplasia, we have mapped the chromosome positions of 5 FBP genes by fluorescence in situ hybridization (FISH) to 10q24 (BTRC alias beta-TRCP/FBW1a), 9q34 (FBXW2 alias FBW2), 13q22 (FBXL3A alias FBL3a), 5p12 (FBXO4 alias FBX4) and 6q25-->q26 (FBXO5 alias FBX5). Since most of these are chromosomal loci frequently altered in tumors, we have screened 42 human tumor cell lines and 48 human tumor samples by Southern hybridization and FISH. While no gross alterations of the genes encoding beta-Trcp/Fbw1a, Fbw2, Fbx4 and Fbx5 were found, heterozygous deletion of the FBXL3A gene was found in four of 13 small cell carcinoma cell lines. This is the first evaluation of genes encoding Fbps in human tumors.

A RA-dependent, tumour-growth suppressive transcription complex is the target of the PML-RARalpha and T18 oncoproteins.

PML and Tif1a are fused to RARA and Braf, respectively, resulting in the production of PML-RARalpha and Tif1alpha-B-Raf (T18) oncoproteins. Here we show that PML, Tif1alpha and RXRalpha/RARalpha function together in a transcription complex that is dependent on retinoic acid (RA). We found that PML acts as a ligand-dependent coactivator of RXRalpha/RARalpha. PML interacts with Tif1alpha and CBP. In Pml-/- cells, the RA-dependent induction of genes such as RARB2 and the ability of Tif1alpha and CBP to act as transcriptional coactivators on RA are impaired. We show that both PML and Tif1alpha are growth suppressors required for the growth-inhibitory activity of RA. T18, similar to PML-RARalpha, disrupts the RA-dependent activity of this complex in a dominant-negative manner resulting in a growth advantage. Our data define a new pathway for the control of cell growth and tumorigenesis, and provide a new model for the pathogenesis of acute promyelocytic leukaemia (APL).

Identification of a family of human F-box proteins.

F-box proteins are an expanding family of eukaryotic proteins characterized by an approximately 40 aminoacid motif, the F box (so named because cyclin F was one of the first proteins in which this motif was identified) [1]. Some F-box proteins have been shown to be critical for the controlled degradation of cellular regulatory proteins [2] [3]. In fact, F-box proteins are one of the four subunits of ubiquitin protein ligases called SCFs. The other three subunits are the Skp1 protein; one of the cullin proteins (Cul1 in metazoans and Cdc53 or Cul A in the yeast Saccharomyces cerevisiae); and the recently identified Roc1 protein (also called Rbx1 or Hrt1). SCF ligases bring ubiquitin conjugating enzymes (either Ubc3 or Ubc4) to substrates that are specifically recruited by the different F-box proteins. The need for high substrate specificity and the large number of known F-box proteins in yeast and worms [2] [4] suggest the existence of a large family of mammalian F-box proteins. Using Skp1 as a bait in a yeast two-hybrid screen and by searching DNA databases, we identified a family of 26 human F-box proteins, 25 of which were novel. Some of these proteins contained WD-40 domains or leucine-rich repeats; others contained either different protein-protein interaction modules or no recognizable motifs. We have named the F-box proteins that contain WD-40 domains Fbws, those containing leucine-rich repeats, Fbls, and the remaining ones Fbxs. We have further characterized representative members of these three classes of F-box proteins.

Critical role played by cyclin D3 in the MyoD-mediated arrest of cell cycle during myoblast differentiation.

During the terminal differentiation of skeletal myoblasts, the activities of myogenic factors regulate not only tissue-specific gene expressions but also the exit from the cell cycle. The induction of cell cycle inhibitors such as p21 and pRb has been shown to play a prominent role in the growth arrest of differentiating myoblasts. Here we report that, at the onset of differentiation, activation by MyoD of the Rb, p21, and cyclin D3 genes occurs in the absence of new protein synthesis and with the requirement of the p300 transcriptional coactivator. In differentiated myocytes, cyclin D3 also becomes stabilized and is found nearly totally complexed with unphosphorylated pRb. The detection of complexes containing cyclin D3, cdk4, p21, and PCNA suggests that cdk4, along with PCNA, may get sequestered into high-order structures held together by pRb and cyclin D3. Cyclin D3 up-regulation and stabilization is inhibited by adenovirus E1A, and this correlates with the ability of E1A to promote pRb phosphorylation; conversely, the overexpression of cyclin D3 in differentiated myotubes counteracts the E1A-mediated reactivation of DNA synthesis. These results indicate that cyclin D3 critically contributes to the irreversible exit of differentiating myoblasts from the cell cycle.

Down-regulation of cyclin B1 gene transcription in terminally differentiated skeletal muscle cells is associated with loss of functional CCAAT-binding NF-Y complex.

The observation that cyclin B1 protein and mRNAs are down-regulated in terminally differentiated (TD) C2C12 cells, suggested us to investigate the transcriptional regulation of the cyclin B1 gene in these cells. Transfections of cyclin B1 promoter constructs indicate that two CCAAT boxes support cyclin B1 promoter activity in proliferating cells. EMSAs demonstrate that both CCAAT boxes are recognized by the trimeric NF-Y complex in proliferating but not in TD cells. Transfecting a dominant-negative mutant of NF-YA we provide evidence that NF-Y is required for maximal promoter activity. Addition of recombinant NF-YA to TD C2C12 nuclear extracts restores binding activity in vitro, thus indicating that the loss of NF-YA in TD cells is responsible for the lack of the NF-Y binding to the CCAAT boxes. Consistent with this, we found that the NF-YA protein is absent in TD C2C12 cells. In conclusion, our data demonstrate that NF-Y is required for cyclin B1 promoter activity. We also demonstrate that cyclin B1 expression is regulated at the transcriptional level in TD C2C12 cells and that the switch-off of cyclin B1 promoter activity in differentiated cells depends upon the loss of a functional NF-Y complex. In particular the loss of NF-YA protein is most likely responsible for its inactivation.

Requirements for measles virus induction of RANTES chemokine in human astrocytoma-derived U373 cells.

Interferons and chemokines play a critical role in regulating the host response to viral infection. Measles virus, a member of the Paramyxoviridae family, induces RANTES expression by astrocytes. We have examined the mechanism of this induction in U373 cells derived from a human astrocytoma. RANTES was induced in a dose- and time-dependent manner by measles virus infection. Inhibition of receptor binding by the anti-CD46 antibody TRA-2.10 and of virus-membrane fusion by the tripeptide X-Phe-Phe-Gly reduced RANTES expression. Formalin-inactivated virus, which can bind but not fuse, and extensively UV-irradiated virus, which can bind and fuse, were both ineffective. Therefore, virus binding to the cellular receptor CD46 and subsequent membrane fusion were necessary, but not sufficient, to induce RANTES. UV irradiation of virus for less than 10 min proportionally inhibited viral transcription and RANTES expression. RANTES induction was decreased in infected cells treated with ribavirin, which inhibits measles virus transcription. However, RANTES mRNA was superinduced by measles virus in the presence of cycloheximide. These data suggest that partial transcription of the viral genome is sufficient and necessary for RANTES induction, whereas viral protein synthesis and replication are not required. This hypothesis was supported by the fact that RANTES was induced through transient expression of the measles virus nucleocapsid gene but not by measles genes encoding P or L proteins or by leader RNA in A549 cells. Thus, transcription of specific portions of measles virus RNA, such as the nucleocapsid gene, appears able to generate the specific signaling required to induce RANTES gene expression.

T cell antigen receptor ubiquitination is a consequence of receptor-mediated tyrosine kinase activation.

Engagement of the T cell antigen receptor results in both its phosphorylation and its ubiquitination. T cell antigen receptor ubiquitination was evaluated in Jurkat, a well characterized human T leukemia cell line. Treatment of cells with the tyrosine kinase inhibitor herbimycin A resulted in an inhibition of receptor ubiquitination. Consistent with this, pervanadate, which increases cellular tyrosine phosphorylation, enhanced receptor ubiquitination. A requirement for receptor-mediated tyrosine kinase activity for ubiquitination was confirmed in cells lacking the tyrosine kinase p56lck and also in cells that are defective in expression of CD45, a tyrosine phosphatase that regulates the activity of p56lck. The need for tyrosine kinase activation for ubiquitination was not bypassed by directly activating protein kinase C and stimulating endocytosis of receptors. These observations establish ubiquitination of the T cell antigen receptor as a tyrosine kinase-dependent manifestation of transmembrane signaling and suggest a role for tyrosine phosphorylation in the ligand-dependent ubiquitination of mammalian transmembrane receptors.

Transcriptional control of the Htf9-A/RanBP-1 gene during the cell cycle.

The mouse Htf9-a gene encodes a small hydrophilic protein, named Ran-binding protein 1 (RanBP-1), for its interaction with the Ras-related Ran protein; RanBP-1 binds the biologically active form of Ran. Ran has been implicated in a variety of nuclear events including DNA replication, RNA export and protein import, and monitoring completion of DNA synthesis before the onset of mitosis; it biological activity is thought to be regulated in S phase. We have investigated whether expression of the Htf9-a/RanBP-1 gene is linked to cell proliferation. Expression of the Htf9-a/RanBP-1 transcript appeared to be dependent on the proliferating state of the cells and peaked in S phase. We have identified a promoter region required for Htf9-a cell cycle-dependent expression and for responsiveness to cell cycle regulators. An E2F-binding site was identified within the cell cycle-regulated promoter region. Expression of the E1A oncoprotein prevented Htf9-a down-regulation in quiescent cells; in addition, both pRb and its relative p107 inhibited transcription from the Htf9-a promoter. These results link the control of Htf9-a/RanBP-1 expression to the cell cycle progression.

MyoD induces retinoblastoma gene expression during myogenic differentiation.

In skeletal muscle cells permanent withdrawal from the cell cycle is a prerequisite for terminal differentiation. The muscle-specific transcription factor MyoD can activate downstream muscle structural genes and myogenic conversion in many different cell types. It has been demonstrated that the product of the retinoblastoma susceptibility gene, with its growth-suppressive activity, is involved in the myogenic function of MyoD (Caruso et al., 1993; Gu et al., 1993). The present study characterises the modulation of retinoblastoma (Rb1) mRNA levels during myogenic differentiation of the murine C2 cell line and provides evidence that the muscle-specific regulatory factor MyoD enhances Rb1 gene transcription. We demonstrate that MyoD mediates the transactivation of a CAT construct whose expression is driven by the human Rb1 gene promoter, and that this is not a consequence of direct binding of MyoD to an E-box DNA sequence motif present in the Rb1 promoter sequences. In addition we have tested the capability of several MyoD mutant proteins of inducing the Rb1 promoter CAT construct. Our results indicate that the MyoD function required for induction of Rb1 promoter activity is distinct from its myogenic function.

Activation-dependent ubiquitination of a T cell antigen receptor subunit on multiple intracellular lysines.

The T cell antigen receptor zeta chain and other T cell antigen receptor components are ubiquitinated on receptor occupancy. A systematic mutagenesis of the zeta subunit was undertaken to determine the sites of ubiquitination. Ubiquitination was found to occur in the cytoplasmic domain of zeta with multiple lysines serving as sites for mono- and polyubiquitination. The mutation of all potential sites of ubiquitination did not inhibit receptor tyrosine phosphorylation or the ubiquitination of other T cell antigen receptor subunits. Lysines introduced into nonnative positions in the zeta molecule were also able to serve as sites for ubiquitination. These findings demonstrate that once a T cell antigen receptor is targeted for ubiquitination, there is little specificity with regard to the lysine residues that are modified.

T cell antigen receptor-eta subunit. Low levels of expression and limited cross-species conservation.

The eta-subunit of the TCR is derived from an alternative splice product of the TCR-zeta gene. The eta-subunit has been extensively characterized in murine T cells, where up to 10% of TCR bear zeta-eta heterodimers rather than zeta-homodimers. In contrast to the significant levels of eta found in murine T cells, in human PBL an eta-like region is expressed spliced to upstream exons of the zeta-gene at no more than 0.25% the level of zeta-mRNA. Analysis of genomic DNA from five additional mammalian species demonstrated that eta-like sequences are highly conserved at a nucleic acid level. The protein sequences encoded in the eta-regions from various species ranged from 28 to 92 amino acids in length, with the first seven deduced amino acids of eta-being common to all species. Within three to five amino acids of this region, all species have five consecutive charged amino acids. Beyond this point, due to translation in different reading frames, there is no significant amino acid homology. The findings of low level of expression of eta-RNA, limited cross-species conservation on a protein level, and the lack of an established functional role for this alternative splice product raise questions as to the potential roles that this subunit may play in TCR function.

Activation-induced ubiquitination of the T cell antigen receptor.

The zeta subunit of the T cell antigen receptor (TCR) exists primarily as a disulfide-linked homodimer. This receptor subunit is important in TCR-mediated signal transduction and is a substrate for a TCR-activated protein tyrosine kinase. The zeta chain was found to undergo ubiquitination in response to receptor engagement. This posttranslational modification occurred in normal T cells and tumor lines. Both nonphosphorylated and phosphorylated zeta molecules were modified, and at least one other TCR subunit, CD3 delta, was also ubiquitinated after activation of the receptor. These findings suggest an expanded role for ubiquitination in transmembrane receptor function.

Mutagenesis of T cell antigen receptor zeta chain tyrosine residues. Effects on tyrosine phosphorylation and lymphokine production.

Occupancy of the T cell antigen receptor triggers a complex set of events that culminate in cellular activation. It is clear that tyrosine kinases play important roles in this process. The zeta subunit of the T cell antigen receptor is a 16-kDa transmembrane structure that exists primarily as a disulfide-linked homodimer. On receptor activation, a subset of zeta molecules undergo tyrosine phosphorylation. To evaluate this process and the role of zeta phosphorylation in T cell activation, site-specific mutagenesis of the intracytoplasmic tyrosines of zeta has been carried out. Analysis of cells expressing these mutant zeta subunits demonstrated that multiple tyrosines underwent phosphorylation in response to receptor engagement, and that the four most carboxyl tyrosines were most crucial to this process. Despite abnormalities in phosphorylation induced by the mutations, lymphokine production in these transfectants was unaffected. Hence, although zeta is a prominent substrate for a receptor-activated tyrosine kinase, neither the mutation of individual tyrosines nor the alteration of the phosphorylation state of the molecule substantively affected the coupling of T cell receptor activation to lymphokine production. These findings raise questions regarding the role of zeta phosphorylation in T cell activation.