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The stress-activated p38 mitogen-activated protein kinase (p38 MAPK), a member of the subgroup of mammalian kinases, appears to play an important role in regulating inflammatory responses, including cytokine secretion and apoptosis. The upstream mediators that link extracellular signals with the p38 MAPK signaling pathway are currently unknown. Here we demonstrate that pp125 focal adhesion kinase-related tyrosine kinase RAFTK (also known as PYK2, CADTK) is activated specifically by methylmethane sulfonate (MMS) and hyperosmolarity but not by ultraviolet radiation, ionizing radiation, or cis-platinum. Overexpression of RAFTK leads to the activation of p38 MAPK. Furthermore, overexpression of a dominant-negative mutant of RAFTK (RAFTK K-M) inhibits MMS-induced p38 MAPK activation. MKK3 and MKK6 are known potential constituents of p38 MAPK signaling pathway, whereas SEK1 and MEK1 are upstream activators of SAPK/JNK and ERK pathways, respectively. We observe that the dominantnegative mutant of MKK3 but not of MKK6, SEK1, or MEK1 inhibits RAFTK-induced p38 MAPK activity. Furthermore, the results demonstrate that treatment of cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid, tetra(acetoxymethyl)-ester, a membranepermeable calcium chelator, inhibits MMS-induced activation of RAFTK and p38 MAPK. Taken together, these findings indicate that RAFTK represents a stress-sensitive mediator of the p38 MAPK signaling pathway in response to certain cytotoxic agents.The mitogen-activated protein kinases (MAPKs) 1 are induced in response to diverse classes of inducers in the transduction of signals from the cell membrane to the nucleus. MAPKs are proline-directed Ser/Thr protein kinases that are regulated by extracellular signals including growth factors and cellular stress (1-3). The well characterized MAPK subfamily includes ERK1 and ERK2, which are activated by growth factors via the conserved Ras/Raf/MEK pathway (4 -7).c-Jun N-terminal protein kinases (JNKs) or stress-activated protein kinases (SAPKs) represent a second class of the mammalian MAPKs, which are primarily activated in response to tumor necrosis factor, interleukin-1, UV-, and DNA-damaging agents (5, 8 -11). A recently identified novel protein tyrosine kinase, related adhesion focal tyrosine kinase (RAFTK) (12) (also known as Pyk2, Refs. 13 and 14); CADTK, Ref. 15) has been shown to be involved upstream to ERKs and JNK signaling pathways (14,16). RAFTK is also a close relative to pp125 FAK tyrosine kinase and is activated by various extracellular signals that increase intracellular calcium concentrations (13). Moreover, RAFTK can tyrosine phosphorylate and modulate the action of ion channels and appears to function as an intermediate that links various calcium signals with both short-and long-term responses in neuronal cells (13).An additional class, which presents substantial similarity to the Saccharomyces cerevisiae HOG1 kinase involved in response to increased extracellular osmolarity (17), is p38 MAPK. p38 MAPK can also be activated by chan...

The caspase family of proteases plays a critical role in the execution of apoptosis. However, efforts to decipher the molecular mechanisms by which caspases induce cell death have been greatly hindered by the lack of systematic and broadly applicable strategies to identify their substrates. Here we describe a novel expression cloning strategy to rapidly isolate cDNAs encoding caspase substrates that are cleaved during apoptosis. Small cDNA pools (approximately 100 clones each) are transcribed/translated in vitro in the presence of [ 35 S]methionine; these labeled protein pools are then incubated with cytosolic extracts from control and apoptotic cells. cDNA pools encoding proteins that are specifically cleaved by the apoptotic extract and whose cleavage is prevented by the caspase inhibitor acetylTyr-Val-Ala-Asp chloromethylketone are subdivided and retested until a single cDNA is isolated. Using this approach, we isolated a partial cDNA encoding protein kinase C-related kinase 2 (PRK2), a serine-threonine kinase, and demonstrate that full-length human PRK2 is proteolyzed by caspase-3 at Asp 117 and Asp 700 in vitro. In addition, PRK2 is cleaved rapidly during Fas-and staurosporine-induced apoptosis in vivo by caspase-3 or a closely related caspase. Both of the major apoptotic cleavage sites of PRK2 in vivo lie within its regulatory domain, suggesting that its activity may be deregulated by proteolysis.Caspases are a novel family of cysteine proteases with aspartate specificity that are related to the Caenorhabditis elegans cell death gene product CED-3. Evidence from many laboratories indicates that caspases play a critical role in the execution of apoptosis. Ectopic expression of these proteases induces programmed cell death. Caspases are normally present in cells as catalytically inactive proenzymes and are proteolytically processed and activated during the induction of apoptosis. Moreover, viral, peptide, and dominant negative inhibitors of caspases delay or prevent programmed cell death (reviewed in Ref. 1). Finally, homozygous inactivation of caspase-1 (2) and caspase-3 (3) in mice results in selective defects in apoptosis.Because caspase activation is a crucial event in apoptosis, it is essential to identify the downstream molecular targets of these proteases whose selective proteolysis is likely to underlie the characteristic morphological features of apoptotic cell death. Although a number of structural and signaling proteins have been shown to be cleaved by caspases during programmed cell death (reviewed in Ref. 4), our understanding of the molecular mechanisms by which caspases induce cell death has been greatly hindered by the lack of systematic and broadly applicable strategies to identify these substrates. Given the absence of such methods and the growing number of caspase family members, it seems likely that the majority of apoptotic caspase targets have yet to be identified.In this report, we describe a method to identify caspase substrates directly and rapidly using labeled protein pools derived...

f RNA-binding proteins participate in a complex array of posttranscriptional controls essential to cell type specification and somatic development. Despite their detailed biochemical characterizations, the degree to which each RNA-binding protein impacts mammalian embryonic development remains incompletely defined, and the level of functional redundancy among subsets of these proteins remains open to question. The poly(C) binding proteins, PCBPs (␣CPs and hnRNP E proteins), are encoded by a highly conserved and broadly expressed gene family. The two major Pcbp isoforms, Pcbp2 and Pcbp1, are robustly expressed in a wide range of tissues and exert both nuclear and cytoplasmic controls over gene expression. Here, we report that Pcbp1-null embryos are rendered nonviable in the peri-implantation stage. In contrast, Pcbp2-null embryos undergo normal development until midgestation (12.5 to 13.5 days postcoitum), at which time they undergo a dramatic loss in viability associated with combined cardiovascular and hematopoietic abnormalities. Mice heterozygous for either Pcbp1 or Pcbp2 null alleles display a mild and nondisruptive defect in initial postpartum weight gain. These data reveal that Pcbp1 and Pcbp2 are individually essential for mouse embryonic development and have distinct impacts on embryonic viability and that Pcpb2 has a nonredundant in vivo role in hematopoiesis. These data further provide direct evidence that Pcbp1, a retrotransposed derivative of Pcpb2, has evolved an essential function(s) in the mammalian genome. P osttranscriptional control of gene expression plays a major role in eukaryotic cell type specification and organism development. RNA processing and mRNA expression are regulated in the nuclear and cytoplasmic compartments by a complex array of interactions among RNA-binding proteins, noncoding RNAs, and target transcripts (1, 2). Studies have documented the central importance of posttranscriptional controls in the programming of embryonic stem cell differentiation and somatic cell development (3-11). The critical role of posttranscriptional control of gene expression can be most clearly recognized in settings of transcriptional inactivity. For example, transcription is globally silenced in the differentiating erythroblast, and the terminal steps in erythrocyte formation are fully dependent on posttranscriptional controls over mRNA stability and translation (12-14). Studies of posttranscriptional controls in this and other models have relied heavily upon a variety of in vitro experimental platforms to define mechanisms and biochemical pathways. While highly informative, such studies do not address the in vivo relevance and nonredundant functions of RNA-binding proteins in physiologically intact environments.The PCBPs (also known as ␣CPs and heterogeneous ribonucleoprotein [hnRNP] E proteins) are a widely expressed and multifunctional family of RNA-binding proteins (15-19) that bind numerous erythroid and nonerythroid mRNAs (20,21). Studies focused on globin gene expression have revealed that t...

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G-quadruplex (G4) sequences are abundant in untranslated regions (UTRs) of human messenger RNAs, but their functional importance remains unclear. By integrating multiple sources of genetic and genomic data, we show that putative G-quadruplex forming sequences (pG4) in 5' and 3' UTRs are selectively constrained, and enriched for cis-eQTLs and RNA-binding protein (RBP) interactions. Using over 15,000 whole-genome sequences, we find that negative selection acting on central guanines of UTR pG4s is comparable to that of missense variation in protein-coding sequences. At multiple GWAS-implicated SNPs within pG4 UTR sequences, we find robust allelic imbalance in gene expression across diverse tissue contexts in GTEx, suggesting that variants affecting G-quadruplex formation within UTRs may also contribute to phenotypic variation. Our results establish UTR G4s as important cis-regulatory elements and point to a link between disruption of UTR pG4 and disease.

Single-cell gene expression analyses of mammalian tissues have uncovered profound stage-specific molecular regulatory phenomena that have changed the understanding of unique cell types and signaling pathways critical for lineage determination, morphogenesis, and growth. We discuss here the case for a Pediatric Cell Atlas as part of the Human Cell Atlas consortium to provide single-cell profiles and spatial characterization of gene expression across human tissues and organs. Such data will complement adult and developmentally focused HCA projects to provide a rich cytogenomic framework for understanding not only pediatric health and disease but also environmental and genetic impacts across the human lifespan.

Ribosome-profiling has uncovered pervasive translation in non-canonical open reading frames, however the biological significance of this phenomenon remains unclear. Using genetic variation from 71,702 human genomes, we assess patterns of selection in translated upstream open reading frames (uORFs) in 5’UTRs. We show that uORF variants introducing new stop codons, or strengthening existing stop codons, are under strong negative selection comparable to protein-coding missense variants. Using these variants, we map and validate gene-disease associations in two independent biobanks containing exome sequencing from 10,900 and 32,268 individuals, respectively, and elucidate their impact on protein expression in human cells. Our results suggest translation disrupting mechanisms relating uORF variation to reduced protein expression, and demonstrate that translation at uORFs is genetically constrained in 50% of human genes.