We describe the discovery of a novel, 20 kDa, secreted human protein named mesencephalic astrocyte-derived neurotrophic factor, or MANF. The homologous, native molecule was initially derived from a rat mesencephalic type-1 astrocyte cell line and recombinant MANF subcloned from a cDNA encoding human arginine-rich protein. MANF selectively protects nigral dopaminergic neurons, versus GABAergic or serotonergic neurons. The discovery of MANF marks a more systematic approach in the search for astrocyte-derived, secreted proteins that selectively protect specific neuronal phenotypes. Compared to glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF), MANF was more selective in the protection of dopaminergic neurons at lower (0.05-0.25 ng/mL) and middle (0.5-2.5 ng/mL) concentrations: MANF>GDNF>BDNF. GDNF was more selective at higher concentrations (25-50 ng/ml): GDNF>MANF>BDNF. Two domains in MANF of 39-AA and 109-AA respectively, and eight cysteines are conserved from C. elegans to man. MANF is encoded by a 4.3 Kb gene with 4 exons, and is located on the short arm of human chromosome 3. The secondary structure is dominated by alpha-helices (47%) and random coils (37%). Studies to determine the localization of MANF in the brains of rat, monkey, and man, as well as the receptor, signaling pathways, and biologically active peptide mimetics are in progress. The selective, neuroprotective effect of MANF for dopaminergic neurons suggests that it may be indicated for the treatment of Parkinson's disease.

In mammalian cells, a specific stress‐activated protein kinase (SAPK/JNK) pathway is activated in response to inflammatory cytokines, injury from heat, chemotherapeutic drugs and UV or ionizing radiation. The mechanisms that link these stimuli to activation of the SAPK/JNK pathway in different tissues remain to be identified. We have developed and applied a PCR‐based subtraction strategy to identify novel genes that are differentially expressed at specific developmental points in hematopoiesis. We show that one such gene, hematopoietic progenitor kinase 1 (hpk1), encodes a serine/threonine kinase sharing similarity with the kinase domain of Ste20. HPK1 specifically activates the SAPK/JNK pathway after transfection into COS1 cells, but does not stimulate the p38/RK or mitogen‐activated ERK signaling pathways. Activation of SAPK requires a functional HPK1 kinase domain and HPK1 signals via the SH3‐containing mixed lineage kinase MLK‐3 and the known SAPK activator SEK1. HPK1 therefore provides an example of a cell type‐specific input into the SAPK/JNK pathway. The developmental specificity of its expression suggests a potential role in hematopoietic lineage decisions and growth regulation.

The c-Src tyrosine kinase phosphorylates and binds to a 68-kDa RNA-binding protein in mitotic cells. We have examined the mechanism and functional consequence of the interaction of c-Src with this protein, Sam 68 (Src associated in mitosis, 68 kDa). In whole cell homogenates, Sam 68 was the predominant substrate and binding partner of overexpressed c-Src. Mitotic, tyrosine-phosphorylated Sam 68 bound selectively to recombinant SH2 domains with significantly different affinities (c-Src approximately Ras GTPase activating protein > p85 alpha (amino-terminal) > Grb2 >> p85 alpha (COOH-terminal)). In vitro translated Sam 68 also bound selectively to recombinant SH3 domains, with the highest affinity for the Src and p85 alpha SH3 domains. SH3 binding was inhibited by specific Sam 68 peptides. In vitro translated Sam 68 bound directly to immobilized poly(U), and this was inhibited by binding of Src and p85 SH3 domains to Sam 68. The results suggest that the selection of Sam 68 as a mitotic target by c-Src is the result of highly specific interaction with SH2 and SH3 domains and that this interaction may modulate the RNA binding activity of Sam 68.

No abstract

Insulin stimulation of Chinese hamster ovary cells expressing the human insulin receptor resulted in a time-dependent decrease in the amount of GTP bound to Rap1. The inactivation of Rap1 was associated with an insulin-stimulated decrease in the amount of Rap1 that was bound to Raf1. In parallel with the dissociation of Raf1 from Rap1, there was an increased association of Raf1 with Ras. Concomitant with the inactivation of Rap1 and decrease in Rap1-Raf1 binding, we observed a rapid insulin-stimulated dissociation of the CrkII-C3G complex which occurred in a Ras-independent manner. The dissociation of the CrkII-C3G was recapitulated in vitro using a GST-C3G fusion protein to precipitate CrkII from whole cell detergent extracts. The association of GST-C3G with CrkII was also dose dependent and demonstrated that insulin reduced the affinity of CrkII for C3G without any effect on CrkII protein levels. Furthermore, the reduction in CrkII binding affinity was reversible by tyrosine dephosphorylation with PTP1B and by mutation of Tyr221 to phenylalanine. Together, these data demonstrate that insulin treatment results in the de-repression of Rap1 inhibitory function on the Raf1 kinase concomitant with Ras activation and stimulation of the downstream Raf1/MEK/ERK cascade.

The dopamine D4 receptor is a G protein-coupled receptor (GPCR) that belongs to the dopamine D2-like receptor family. Functionally, the D2-like receptors are characterized by their ability to inhibit adenylyl cyclase. The dopamine D4 receptor as well as many other catecholaminergic receptors contain several putative SH3 binding domains. Most of these sites in the D4 receptor are located in a polymorphic repeat sequence and flanking sequences in the third intracellular loop. Here we demonstrate that this region of the D4 receptor can interact with a large variety of SH3 domains of different origin. The strongest interactions were seen with the SH2−SH3 adapter proteins Grb2 and Nck. The repeat sequence itself is not essential in this interaction. The data presented indicate that the different SH3 domains in the adapter proteins interact in a cooperative fashion with two distinct sites immediately upstream and downstream from the repeat sequence. Removal of all the putative SH3 binding domains in the third intracellular loop of the dopamine D4 receptor resulted in a receptor that could still bind spiperone and dopamine. Dopamine could not modulate the coupling of these mutant receptors to adenylyl cyclase and MAPK, although dopamine modulated receptor−G protein interaction appeared normal. The receptor deletion mutants show strong constitutive internalization that may account for the deficiency in functional activation of second messengers. The data indicates that the D4 receptor contains SH3 binding sites and that these sites fall within a region involved in the control of receptor internalization.

The Src homology 2 (SH2) domain of the mammalian adaptor protein Crk-II contains a proline-rich insert, predicted to lie within an extended DE loop, which is dispensable for phosphopeptide binding. Using the yeast two-hybrid system, this region of the Crk-II SH2 domain was found to interact with a subset of SH3 domains, notably the Abl SH3 domain. Furthermore, this proline-rich insert was found to modify the efficiency with which Crk-II was phosphorylated by the p140(c-abl) tyrosine kinase. In vitro, the interaction of full-length non-phosphorylated Crk-II with a glutathione S-transferase-Abl SH3 domain fusion protein was very weak. However, phosphorylation of Crk-II on Tyr-221 which induces an intramolecular association with the SH2 domain, or addition of a phosphopeptide corresponding to the Crk-II Tyr-221 phosphorylation site, stimulated association of Crk-II with the Abl SH3 domain. NMR spectroscopic analysis showed that binding of the Tyr-221 phosphopeptide to the Crk SH2 domain induced a chemical shift change in Val-71, located in the proline-rich insert, indicative of a change in the structure of the proline-rich loop in response of Crk SH2-pTyr-221 interaction. These results suggest that the proline-rich insert in the Crk SH2 domain constitutes an SH3 domain-binding site that can be regulated by binding of a phosphopeptide ligand to the Crk SH2 domain.

We report on the potency of two Tyrphostin tyrosine kinase blockers, AG 1112 and AG 568, to inhibit p210bcr-abl tyrosine kinase activity in K562 cells, concomitant with the induction of erythroid differentiation. AG 568 and especially AG 1112 represent a specific group of nontoxic protein tyrosine kinase blockers among more than 1,400 tested. These compounds possess therapeutic potential for purging Philadelphia chromosome-positive cells in preparation for autologous bone marrow transplantation in chronic myelogenous leukemia.