ABSTRACT
Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a molecular target for the sensitization of cancer cells to the FDA-approved topoisomerase inhibitors topotecan and irinotecan. High-throughput screening of natural product extract and fraction libraries for inhibitors of TDP1 activity resulted in the discovery of a new class of knotted cyclic peptides from the marine sponge Axinella sp. Bioassay-guided fractionation of the source extract resulted in the isolation of the active component which was determined to be an unprecedented 42-residue cysteine-rich peptide named recifin A. The native NMR structure revealed a novel fold comprising a four strand antiparallel ß-sheet and two helical turns stabilized by a complex disulfide bond network that creates an embedded ring around one of the strands. The resulting structure, which we have termed the Tyr-lock peptide family, is stabilized by a tyrosine residue locked into three-dimensional space. Recifin A inhibited the cleavage of phosphodiester bonds by TDP1 in a FRET assay with an IC50 of 190 nM. Enzyme kinetics studies revealed that recifin A can specifically modulate the enzymatic activity of full-length TDP1 while not affecting the activity of a truncated catalytic domain of TDP1 lacking the N-terminal regulatory domain (Δ1-147), suggesting an allosteric binding site for recifin A on the regulatory domain of TDP1. Recifin A represents both the first of a unique structural class of knotted disulfide-rich peptides and defines a previously unseen mechanism of TDP1 inhibition that could be productively exploited for potential anticancer applications.
Subject(s)
Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Peptides/chemistry , Peptides/pharmacology , Phosphoric Diester Hydrolases/metabolism , Tyrosine , Allosteric Regulation/drug effects , Amino Acid Sequence , Catalytic Domain , Disulfides/chemistry , High-Throughput Screening Assays , Phosphoric Diester Hydrolases/chemistryABSTRACT
Cyclin-dependent kinase 5 (Cdk5) is predominantly expressed in the nervous system, where it is involved in neuronal migration, synaptic transmission, and survival. The role of Cdk5 in synaptic transmission is mediated by regulating the cellular functions of presynaptic proteins such as synapsin, Munc18, and dynamin 1. Its multifunctional role at the synapse is complex and probably involves other novel substrates. To explore this possibility, we used a yeast two-hybrid screen of a human cDNA library with p35 as bait and isolated human septin 5 (SEPT5), known also as hCDCrel-1, as an interacting clone. Here we report that p35 associates with SEPT5 in GST (glutathione S-transferase)-pull-down and coimmunoprecipitation assays. We confirmed that Cdk5/p35 phosphorylates SEPT5 in vitro and in vivo and identified S327 of SEPT5 as a major phosphorylation site. A serine (S)-to-alanine (A) 327 mutant of SEPT5 bound syntaxin more efficiently than SEPT5 wild type. Additionally, coimmunoprecipitation from synaptic vesicle fractions and Cdk5 wild-type and knock-out lysates showed that phosphorylation of septin 5 by Cdk5/p35 decreases its binding to syntaxin-1. Moreover, mutant nonphosphorylated SEPT5 potentiated regulated exocytosis more than the wild type when each was expressed in PC12 cells. These data suggest that Cdk5 phosphorylation of human septin SEPT5 at S327 plays a role in modulating exocytotic secretion.
Subject(s)
Cell Cycle Proteins/metabolism , Cyclin-Dependent Kinase 5/physiology , Exocytosis/physiology , Neurons/physiology , Animals , Binding Sites/physiology , Brain/cytology , Brain/metabolism , Cells, Cultured , Cricetinae , Cricetulus , Embryo, Mammalian , Growth Hormone/metabolism , Humans , Immunoprecipitation/methods , Mutation/physiology , Nerve Tissue Proteins/metabolism , Neurons/drug effects , Phosphorylation , Protein Binding , Protein Structure, Tertiary/physiology , RNA, Small Interfering/metabolism , Rats , SNARE Proteins/metabolism , Septins , Serine/metabolism , Synaptic Vesicles/metabolism , Syntaxin 1/metabolism , Transfection/methodsABSTRACT
Celecoxib, a selective inhibitor of cyclooxygenase-2 (Cox-2), was efficacious in clinical prevention trials of patients with familial adenomatous polyposis (FAP) and sporadic colorectal cancer. To identify as yet poorly defined molecular determinants of celecoxib efficacy, a multidimensional serum fractionation approach was used coupled with nanospray tandem mass spectrometry to perform label-free global proteomic profiling of serum samples from the FAP/celecoxib prevention trial. Subsequently, the application of an algorithm for large-scale biomarker discovery on comparative serum proteomic profiles of pre- and post-celecoxib treatment samples identified 83 potentially celecoxib-responsive proteins from various cellular compartments, biological processes and molecular functions. Celecoxib modulation of some of these proteins was confirmed in serum samples of FAP patients and colorectal cancer cell lines by Western blotting. Thus, using a shotgun procedure to rapidly identify important celecoxib-modulated proteins, this pilot study has uncovered novel systemic changes some of which are highly relevant for carcinogenesis and vascular biology. Validation of selected markers, especially those involved in key signaling networks and those considered molecular indicators of cardiovascular pathology, in larger celecoxib clinical trials is expected to provide insights into the molecular mechanisms of celecoxib and the efficacy/toxicity issues related to its use as a chemopreventive agent.
Subject(s)
Adenomatous Polyposis Coli/drug therapy , Adenomatous Polyposis Coli/metabolism , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Blood Proteins/metabolism , Proteomics , Pyrazoles/pharmacology , Sulfonamides/pharmacology , Adenomatous Polyposis Coli/pathology , Blotting, Western , Celecoxib , Chromatography, Liquid , Computational Biology , Humans , Peptide Fragments , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
Parafibromin, the product of the HRPT2 (hyperparathyroidism-jaw tumor syndrome 2) tumor suppressor gene, is the human homologue of yeast Cdc73, part of the yeast RNA polymerase II/Paf1 complex known to be important for histone modification and connections to posttranscriptional events. By purifying cellular parafibromin and characterizing its associated proteins, we have identified a human counterpart to the yeast Paf1 complex including homologs of Leo1, Paf1, and Ctr9. Like the yeast complex, the parafibromin complex associates with the nonphosphorylated and Ser2 and Ser5 phosphorylated forms of the RNA polymerase II large subunit. Immunofluorescence experiments show that parafibromin is a nuclear protein. In addition, cotransfection data suggest that parafibromin can interact with a histone methyltransferase complex that methylates histone H3 on lysine 4. Some mutant forms of parafibromin lack association with hPaf1 complex members and with the histone methyltransferase complex, suggesting that disruption of these complexes may correlate with the oncogenic process.
Subject(s)
Multiprotein Complexes , Proteins/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line , Histone Methyltransferases , Histone-Lysine N-Methyltransferase/metabolism , Humans , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Protein Methyltransferases , Proteins/genetics , RNA Polymerase II/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Transcriptional Elongation Factors , Tumor Suppressor ProteinsABSTRACT
IL-7 is required for T cell differentiation and mature T cell homeostasis and promotes pro-B cell proliferation and survival. Tyrosine phosphorylation plays a central role in IL-7 signaling. We identified by two-dimensional electrophoresis followed by anti-phosphotyrosine immunoblotting and mass spectrometry sixteen tyrosine phosphorylated proteins from the IL-7-dependent cell line D1. IL-7 stimulation induced the phosphorylation of the proteins STI1, ATIC and hnRNPH, involved in pathways related to survival, proliferation and gene expression, respectively, and increased the phosphorylation of CrkL, a member of a family of adaptors including the highly homologous Crk isoforms CrkII and CrkI, important in multiple signaling pathways. We observed an increased phosphorylation of CrkL in murine pro-B cells and in murine and human T cells. In addition, IL-7 increased the association of CrkL with the transcription factor Stat5, essential for IL-7 pro-survival activity. The selective tyrosine kinase inhibitor Imatinib. counteracted the IL-7 pro-survival effect in D1 cells and decreased CrkL phosphorylation. These data suggested that CrkL could play a pro-survival role in IL-7-mediated signaling. We observed that pro-B cells also expressed, in addition to CrkL, the Crk isoforms CrkII and CrkI and therefore utilized pro-B cells conditionally deficient in all three to evaluate the role of these proteins. The observation that the IL-7 pro-survival effect was reduced in Crk/CrkL conditionally-deficient pro-B cells further pointed to a pro-survival role of these adaptors. To further evaluate the role of these proteins, gene expression studies were performed in Crk/CrkL conditionally-deficient pro-B cells. IL-7 decreased the transcription of the receptor LAIR1, which inhibits B cell proliferation, in a Crk/CrkL-dependent manner, suggesting that the Crk family of proteins may promote pro-B cell proliferation. Our data contribute to the understanding of IL-7 signaling and suggest the involvement of Crk family proteins in pathways promoting survival and proliferation.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Interleukin-7/pharmacology , Nuclear Proteins/metabolism , Adaptor Proteins, Signal Transducing/genetics , Animals , Apoptosis/drug effects , B-Lymphocytes/cytology , B-Lymphocytes/drug effects , B-Lymphocytes/metabolism , Gene Expression Regulation/drug effects , Humans , Imatinib Mesylate/pharmacology , Male , Mice , Mice, Inbred C57BL , Nuclear Proteins/genetics , Phosphorylation/drug effects , Protein Isoforms/genetics , Protein Isoforms/metabolism , STAT5 Transcription Factor/metabolism , Signal Transduction/drug effects , T-Lymphocytes/cytology , T-Lymphocytes/drug effects , T-Lymphocytes/metabolismABSTRACT
The epigenetic programming of genomic DNA is accomplished, in part, by several DNA cytosine-5-methyltransferases that act by covalently modifying cytosines with the addition of a methyl group. This covalent modification is maintained by the DNA cytosine-5-methyltransferase-1 enzyme (DNMT1), which is capable of acting in concert with other similar enzymes to silence important tumor suppressor genes. IL-6 is a multifunctional mediator of inflammation, acting through several major signaling cascades, including the phosphatidylinositol-3-kinase pathway (PI-3-K), which activates protein kinase B (AKT/PKB) downstream. Here, we show that the subcellular localization of DNMT1 can be altered by the addition of IL-6, increasing the rate of nuclear translocation of the enzyme from the cytosolic compartment. The mechanism of nuclear translocation of DNMT1 is greatly enhanced by phosphorylation of the DNMT1 nuclear localization signal (NLS) by PKB/AKT kinase. Mutagenic alteration of the two AKT target amino acids within the NLS results in a major loss of DNMT1 nuclear translocation, while the creation of a "phospho-mimic" amino acid (mutation to acidic residues) restores this compartmentation ability. These observations suggest an interesting hypothesis regarding how mediators of chronic inflammation may disturb the delicate balance of cellular compartmentalization of important proteins, and reveals a potential mechanism for the induction or enhancement of tumor growth via alteration of the components involved in the epigenetic programming of a cell.
Subject(s)
Cell Nucleus/drug effects , Cell Nucleus/enzymology , DNA (Cytosine-5-)-Methyltransferases/metabolism , Interleukin-6/pharmacology , Nuclear Localization Signals/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Amino Acid Sequence , Cell Line , DNA (Cytosine-5-)-Methyltransferase 1 , DNA (Cytosine-5-)-Methyltransferases/chemistry , Green Fluorescent Proteins/metabolism , Humans , Isotope Labeling , Karyopherins/metabolism , Molecular Sequence Data , Mutant Proteins/metabolism , Phosphorus Radioisotopes , Phosphorylation/drug effects , Protein Binding/drug effects , Protein Structure, Tertiary , Protein Transport/drug effects , Recombinant Fusion Proteins/metabolism , Sequence Analysis, ProteinABSTRACT
Mitogen-activated protein kinase (MAPK) p38 is part of a broad and ubiquitously expressed family of MAPKs whose activity is responsible for mediating an intracellular response to extracellular stimuli through a phosphorylation cascade. p38 is central to this signaling node and is activated by upstream kinases while being responsible for activating downstream kinases and transcription factors via phosphorylation. Dysregulated p38 activity is associated with numerous autoimmune disorders and has been implicated in the progression of several types of cancer. A number of p38 inhibitors have been tested in clinical trials, with none receiving regulatory approval. One characteristic shared by all of the compounds that failed clinical trials is that they are all adenosine triphosphate (ATP)-competitive p38 inhibitors. Seeing this lack of mechanistic diversity as an opportunity, we screened ~32,000 substances in search of novel p38 inhibitors. Among the inhibitors discovered is a compound that is both non-ATP competitive and biologically active in cell-based models for p38 activity. This is the first reported discovery of a non-ATP-competitive p38 inhibitor that is active in cells and, as such, may enable new pharmacophore designs for both therapeutic and basic research to better understand and exploit non-ATP-competitive inhibitors of p38 activity.
Subject(s)
Drug Discovery/methods , Protein Kinase Inhibitors/pharmacology , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , Adenosine Triphosphate/metabolism , Antineoplastic Agents/pharmacology , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Cell Line, Tumor , Cytokines/metabolism , Enzyme-Linked Immunosorbent Assay/methods , High-Throughput Screening Assays , Humans , Inhibitory Concentration 50 , Protein Binding , Recombinant Fusion Proteins , Small Molecule Libraries , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
The highly conserved, atypical RIO serine protein kinases are found in all organisms, from archaea to man. In yeast, the kinase activity of Rio2 is necessary for the final processing step of maturing the 18S ribosomal rRNA. We have previously shown that the Rio2 protein from Archaeoglobus fulgidus contains both a small kinase domain and an N-terminal winged helix domain. Previously solved structures using crystals soaked in nucleotides and Mg2+ or Mn2+ showed bound nucleotide but no ordered metal ions, leading us to the conclusion that they did not represent an active conformation of the enzyme. To determine the functional form of Rio2, we crystallized it after incubation with ATP or ADP and Mn2+. Co-crystal structures of Rio2-ATP-Mn and Rio2-ADP-Mn were solved at 1.84 and 1.75 angstroms resolution, respectively. The gamma-phosphate of ATP is firmly positioned in a manner clearly distinct from its location in canonical serine kinases. Comparison of the Rio2-ATP-Mn complex with the Rio2 structure with no added nucleotides and with the ADP complex indicates that a flexible portion of the Rio2 molecule becomes ordered through direct interaction between His126 and the gamma-phosphate oxygen of ATP. Phosphopeptide mapping of the autophosphorylation site of Rio2 identified Ser128, within the flexible loop and directly adjacent to the part that becomes ordered in response to ATP, as the target. These results give us further information about the nature of the active site of Rio2 kinase and suggest a mechanism of regulation of its enzymatic activity.
Subject(s)
Archaeal Proteins/chemistry , Archaeal Proteins/metabolism , Archaeoglobus fulgidus/enzymology , Manganese/metabolism , Protein Conformation , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/metabolism , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Archaeal Proteins/genetics , Binding Sites , Crystallization , Crystallography, X-Ray , Histidine/chemistry , Histidine/metabolism , Models, Molecular , Molecular Sequence Data , Phosphorylation , Protein Serine-Threonine Kinases/genetics , Sequence Homology, Amino Acid , Serine/chemistry , Serine/metabolismABSTRACT
Rio1 is the founding member of the RIO family of atypical serine kinases that are universally present in all organisms from archaea to mammals. Activity of Rio1 was shown to be absolutely essential in Saccharomyces cerevisiae for the processing of 18S ribosomal RNA, as well as for proper cell cycle progression and chromosome maintenance. We determined high-resolution crystal structures of Archaeoglobus fulgidus Rio1 in the presence and absence of bound nucleotides. Crystallization of Rio1 in the presence of ATP or ADP and manganese ions demonstrated major conformational changes in the active site, compared with the uncomplexed protein. Comparisons of the structure of Rio1 with the previously determined structure of the Rio2 kinase defined the minimal RIO domain and the distinct features of the RIO subfamilies. We report here that Ser108 represents the sole autophosphorylation site of A. fulgidus Rio1 and have therefore established its putative peptide substrate. In addition, we show that a mutant enzyme that cannot be autophosphorylated can still phosphorylate an inactive form of Rio1, as well as a number of typical kinase substrates.
Subject(s)
Archaeal Proteins/chemistry , Archaeal Proteins/metabolism , Archaeoglobus fulgidus/enzymology , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray , Humans , Models, Molecular , Molecular Sequence Data , Nucleotides/chemistry , Nucleotides/metabolism , Phosphorylation , Protein Structure, Tertiary , Sequence AlignmentABSTRACT
Interleukin-7 (IL-7) is critical for T-cell development and peripheral T-cell homeostasis. The survival of pro-T cells and mature T cells requires IL-7. The survival function of IL-7 is accomplished partly through induction of the antiapoptotic protein Bcl-2 and inhibition of proapoptotic proteins Bax and Bad. We show here that the proapoptotic protein Bim, a BH3-only protein belonging to the Bcl-2 family, also plays a role in peripheral T-cell survival. Deletion of Bim partially protected an IL-7-dependent T-cell line and peripheral T cells, especially cells with an effector memory phenotype, from IL-7 deprivation. However, T-cell development in the thymus was not restored in IL-7(-/-) Rag2(-/-) mice reconstituted with Bim(-/-) bone marrow. IL-7 withdrawal altered neither the intracellular location of Bim, which was constitutively mitochondrial, nor its association with Bcl-2; however, a reduction in its association with the prosurvival protein Mcl-1 was observed. IL-7 withdrawal did not increase Bim mRNA or protein expression but did induce changes in the isoelectric point of Bim(EL) and its reactivity with an antiphosphoserine antibody. Our findings suggest that the maintenance of peripheral T cells by IL-7 occurs partly through inhibition of Bim activity at the posttranslational level.
Subject(s)
Apoptosis Regulatory Proteins/metabolism , Apoptosis/immunology , DNA-Binding Proteins/metabolism , Interleukin-7/metabolism , Membrane Proteins/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Proto-Oncogene Proteins/metabolism , T-Lymphocytes/immunology , Adoptive Transfer , Animals , Apoptosis Regulatory Proteins/genetics , Bcl-2-Like Protein 11 , Cell Line , Cell Survival/physiology , DNA-Binding Proteins/genetics , Interleukin-7/genetics , Membrane Proteins/genetics , Mice , Mice, Inbred C57BL , Myeloid Cell Leukemia Sequence 1 Protein , Proto-Oncogene Proteins/genetics , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
The bZIP transcription factor C/EBPbeta is a target of Ras signaling that has been implicated in Ras-induced transformation and oncogene-induced senescence (OIS). To gain insights into Ras-C/EBPbeta signaling, we investigated C/EBPbeta activation by oncogenic Ras. We show that C/EBPbeta DNA binding is autorepressed and becomes activated by the Ras-Raf-MEK-ERK-p90(RSK) cascade. Inducible phosphorylation by RSK on Ser273 in the leucine zipper was required for DNA binding. In addition, three other modifications (phosphorylation on Tyr109 [p-Tyr109], p-Ser111, and monomethylation of Arg114 [me-Arg114]) within an N-terminal autoinhibitory domain were important for Ras-induced C/EBPbeta activation and cytostatic activity. Apart from its role in DNA binding, Ser273 phosphorylation also creates an interhelical g<-->e' salt bridge with Lys268 that increases attractive electrostatic interactions between paired leucine zippers and promotes homodimerization. Mutating Ser273 to Ala or Lys268 to Glu decreased C/EBPbeta homodimer formation, whereas heterodimerization with C/EBPgamma was relatively unaffected. The S273A substitution also reduced the antiproliferative activity of C/EBPbeta in Ras(V12)-expressing fibroblasts and decreased binding to target cell cycle genes, while a phosphomimetic substitution (S273D) maintained growth arrest function. Our findings identify four novel C/EBPbeta-activating modifications, including RSK-mediated phosphorylation of a bifunctional residue in the leucine zipper that regulates DNA binding and homodimerization and thereby promotes cell cycle arrest.
Subject(s)
CCAAT-Enhancer-Binding Protein-beta/metabolism , Cell Cycle/physiology , DNA/metabolism , Leucine Zippers , Ribosomal Protein S6 Kinases/metabolism , Amino Acid Sequence , Animals , CCAAT-Enhancer-Binding Protein-beta/chemistry , CCAAT-Enhancer-Binding Protein-beta/genetics , Cell Line , DNA/genetics , Growth Substances/metabolism , Humans , MAP Kinase Signaling System/physiology , Mice , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinases/genetics , Mitogen-Activated Protein Kinases/metabolism , Molecular Sequence Data , Phosphorylation , Protein Multimerization , Protein Processing, Post-Translational , Rats , Ribosomal Protein S6 Kinases/antagonists & inhibitors , Ribosomal Protein S6 Kinases/genetics , Sequence Alignment , Transcriptional Activation , ras Proteins/genetics , ras Proteins/metabolismABSTRACT
PTIP, a protein with tandem BRCT domains, has been implicated in DNA damage response. However, its normal cellular functions remain unclear. Here we show that while ectopically expressed PTIP is capable of interacting with DNA damage response proteins including 53BP1, endogenous PTIP, and a novel protein PA1 are both components of a Set1-like histone methyltransferase (HMT) complex that also contains ASH2L, RBBP5, WDR5, hDPY-30, NCOA6, SET domain-containing HMTs MLL3 and MLL4, and substoichiometric amount of JmjC domain-containing putative histone demethylase UTX. PTIP complex carries robust HMT activity and specifically methylates lysine 4 (K4) on histone H3. Furthermore, PA1 binds PTIP directly and requires PTIP for interaction with the rest of the complex. Moreover, we show that hDPY-30 binds ASH2L directly. The evolutionarily conserved hDPY-30, ASH2L, RBBP5, and WDR5 likely constitute a subcomplex that is shared by all human Set1-like HMT complexes. In contrast, PTIP, PA1, and UTX specifically associate with the PTIP complex. Thus, in cells without DNA damage agent treatment, the endogenous PTIP associates with a Set1-like HMT complex of unique subunit composition. As histone H3 K4 methylation associates with active genes, our study suggests a potential role of PTIP in the regulation of gene expression.
Subject(s)
Carrier Proteins/metabolism , DNA-Binding Proteins/metabolism , Histone-Lysine N-Methyltransferase/metabolism , Histones/metabolism , Multiprotein Complexes/metabolism , Nuclear Proteins/metabolism , Protein Processing, Post-Translational/physiology , Animals , Carrier Proteins/genetics , Cell Line, Transformed , DNA Damage/genetics , DNA-Binding Proteins/genetics , HeLa Cells , Histone Methyltransferases , Histone-Lysine N-Methyltransferase/genetics , Histones/genetics , Humans , Lysine/genetics , Lysine/metabolism , Methylation , Mice , Mice, Knockout , Multiprotein Complexes/genetics , Nuclear Proteins/genetics , Protein MethyltransferasesABSTRACT
Signaling-responsive MAP kinases (MAPKs) are key in mediating immune responses and are activated through the phosphorylation of a Thr-X-Tyr motif by upstream MAPK kinases. Here we show that T cells stimulated through the T cell receptor (TCR) used an alternative mechanism in which p38 was phosphorylated on Tyr323 and subsequently autophosphorylated residues Thr180 and Tyr182. This required the TCR-proximal tyrosine kinase Zap70 but not the adaptor protein LAT, which was required for activation of extracellular signal-regulated protein kinase MAPKs. TCR activation of p38 lacking Tyr323 was diminished, and blocking of p38 activity prevented p38 dual phosphorylation in normal T cells but not in B cells. Thus, phosphorylation of Tyr323 dependent on the tyrosine kinase Lck and mediated by Zap70 serves as an important mechanism for TCR activation of p38 in T cells.