Sam68, the 68-kDa Src substrate associated during mitosis, is an RNA-binding protein with signaling properties that contains a GSG (GRP33, Sam68, GLD-1) domain. Here we report the cloning of two Sam68-likemammalian proteins, SLM-1 and SLM-2. These proteins have an Ϸ70% sequence identity with Sam68 in their GSG domain. SLM-1 and SLM-2 have the characteristic Sam68 SH2 and SH3 domain binding sites. SLM-1 is an RNA-binding protein that is tyrosine phosphorylated by Src during mitosis. SLM-1 bound the SH2 and SH3 domains of p59 fyn , Grb-2, phospholipase C␥-1 (PLC␥-1), and͞or p120 rasGAP , suggesting it may function as a multifunctional adapter protein for Src during mitosis. SLM-2 is an RNA-binding protein that is not tyrosine phosphorylated by Src or p59 fyn . Moreover, SLM-2 did not associate with the SH3 domains of p59 fyn , Grb-2, PLC␥-1, or p120 rasGAP , suggesting that SLM-2 may not function as an adapter protein for these proteins. The identification of SLM-1 and SLM-2 demonstrates the presence of a Sam68͞SLM family whose members have the potential to link signaling pathways with RNA metabolism.

The role of arginine methylation in Drosophila melanogaster is unknown. We identified a family of nine PRMTs (protein arginine methyltransferases) by sequence homology with mammalian arginine methyltransferases, which we have named DART1 to DART9 ( Drosophila arginine methyltransferases 1-9). In keeping with the mammalian PRMT nomenclature, DART1, DART4, DART5 and DART7 are the putative homologues of PRMT1, PRMT4, PRMT5 and PRMT7. Other DART family members have a closer resemblance to PRMT1, but do not have identifiable homologues. All nine genes are expressed in Drosophila at various developmental stages. DART1 and DART4 have arginine methyltransferase activity towards substrates, including histones and RNA-binding proteins. Amino acid analysis of the methylated arginine residues confirmed that both DART1 and DART4 catalyse the formation of asymmetrical dimethylated arginine residues and they are type I arginine methyltransferases. The presence of PRMTs in D. melanogaster suggest that flies are a suitable genetic system to study arginine methylation.

Dual leucine zipper-bearing kinase (DLK) is a mixed-lineage kinase family member that acts as an upstream activator of the c-Jun N-terminal kinases. As opposed to other components of this pathway, very little is currently known regarding the mechanisms by which DLK is regulated in mammalian cells. Here we identify the stress-inducible heat shock protein 70 (Hsp70) as a negative regulator of DLK expression and activity. Support for this notion derives from data showing that Hsp70 induces the proteasomal degradation of DLK when both proteins are coexpressed in COS-7 cells. Hsp70-mediated degradation occurs with expression of wild-type DLK, which functions as a constitutively activated protein in these cells but not kinase-defective DLK. Interestingly, the Hsp70 co-chaperone CHIP, an E3 ubiquitin ligase, seems to be indispensable for this process since Hsp70 failed to induce DLK degradation in COS-7 cells expressing a CHIP mutant unable to catalyze ubiquitination or in immortalized fibroblasts derived from CHIP knock-out mice. Consistent with these data, we have found that endogenous DLK becomes sensitive to CHIP-dependent proteasomal degradation when it is activated by okadaic acid and that down-regulation of Hsp70 levels with an Hsp70 antisense attenuates this sensitivity. Therefore, our studies suggest that Hsp70 contributes to the regulation of activated DLK by promoting its CHIPdependent proteasomal degradation.Dual leucine zipper-bearing kinase (DLK) 2 is a serine/threonine kinase that belongs to a family of mitogen-activated protein kinase kinase kinases, known as mixed-lineage kinases (MLKs) (1). Members of this family, which also include MLK1, MLK2, MLK3, MLK4, leucine zipper-bearing kinase, and leucine zipper and sterile ␣-motif kinase (1), are characterized at the structural level by the presence of a catalytic domain bearing amino acid motifs found in serine/threonine and tyrosine kinases and one or two leucine zipper motifs, which regulate their activity by mediating protein dimerization or oligomerization (2-5). A number of other interesting motifs that are likely important for protein binding have also been identified in specific members of the MLK family. For instance, MLK2 and MLK3 contain a Src homology 3 (SH3) domain in their N-terminal region that binds, respectively, the GTPase dynamin and the Ste20-related protein kinase HPK1 (6, 7). Both MLK proteins also possess a functional Cdc42/Rac interactive binding (CRIB) motif that mediates association with Cdc42 and Rac1 in a GTP-dependent manner (8, 9).The importance of the MLKs as signaling molecules is highlighted by the fact that these proteins act as key regulators of the c-Jun N-terminal kinase (JNK) subgroup of mitogen-activated protein kinases (1). Specifically, all MLK family members regulate the JNK pathway by phosphorylating and activating the JNK direct upstream activators . In addition to their role in catalyzing JNK activation, MLKs are also known to contribute to apoptosis in neuronal cells. Indeed, when dominant negative forms of MLKs...

The Mpv 20 transgenic mouse strain was created by infection of embryos with a defective retrovirus. When Mpv 20 heterozygous animals were crossed, no homozygous neonatal mice or midgestation embryos were identified. When embryos from heterozygous crosses were cultured in vitro, approximately one quarter arrested as uncompacted eight-cell embryos, indicating that proviral insertion resulted in a recessive lethal defect whose phenotype was manifest very early in development. Molecular cloning of the Mpv 20 insertion site revealed that the provirus had disrupted the Npat gene, a gene of unknown function, resulting in the production of a truncated Npat mRNA. Expression of the closely linked Atm gene was found to be unaffected by the provirus.The insertion of retroviral proviruses into the host genome can have mutagenic consequences, either by the activation of host genes through juxtaposition of positive viral regulatory elements (7) or by the transcriptional silencing of adjacent host sequences that occurs as murine type C viruses infect germ line cells (15,16). The former phenomenon has been exploited with great success for the discovery of oncogenes; the latter has been useful for identifying mammalian developmental mutations (8, 21). Through retroviral insertional mutagenesis screens, genes whose inactivation caused recessive lethal defects manifest at the midgestation (25), neonatal (22), and adult stages (29) have been identified. The use of defective viruses incorporating selectable markers has further enhanced the utility of the strategy by allowing the infection and selection of ES cell clones in which genes have been trapped (3, 5). There are two great advantages in using retroviruses as mutagenic agents: they generate discrete, localized insertion mutations (as opposed to the large deletions that frequently accompany the insertion of DNA introduced by pronuclear microinjection), and the provirus provides the probe with which the site of insertion can be cloned.We have used the defective murine type C virus myeloproliferative sarcoma virus (MPSV)-neo (23) to infect early mouse embryos in a screen which previously gave rise to the adult kidney-specific mutant strain Mpv 17 (29). In this paper, we describe Mpv 20, a mouse strain in which insertional mutagenesis has resulted in a recessive lethal phenotype manifest at a very early stage of embryonic development. We also report the identification of the mutated gene in Mpv 20 as Npat, a recently described gene closely linked to the Atm locus (13). No function has been reported for Npat, and its predicted amino acid sequence is not sufficiently similar to known sequences to allow predictions about the function of the Npat protein. The data presented herein suggest that whatever its function, the Npat gene protein is absolutely required for development beyond the eight-cell stage. MATERIALS AND METHODS Generation of transgenic mouse lines.Retroviral infection of 4-to 16-cell CFW mouse embryos was accomplished as described previously (15, 29). Briefly, Rat 1 cell...