Cytokinesis and septation in the fission yeast Schizosaccharomyces pombe are studied as a model for mammalian cell division. In fission yeast, septation is positively regulated by Spg1, a Ras family GTPase that localizes to spindle-pole bodies (SPBs) throughout the cell cycle. As cells enter mitosis, Spg1 accumulates in an active, GTPbound form and binds the Cdc7 protein kinase to cause Cdc7 translocation to SPBs. Cdc7 disappears from one SPB in mid-anaphase and from the second SPB in late mitosis. Byr4 plus Cdc16 negatively regulate septation by forming a two-component GTPase-activating protein for Spg1. These results led us to hypothesize that Byr4 localization to SPBs regulated the nucleotide state of Spg1, due to its ability to form Spg1GAP activity with Cdc16 and thus the binding of Cdc7 to Spg1 at SPBs. To test this hypothesis, Byr4 localization was determined using indirect immunofluorescence. This analysis revealed that Byr4 was localized to SPBs that did not contain Cdc7. In byr4 ؊ mutants, Cdc7 localized to interphase SPBs and only symmetrically localized to mitotic SPBs. In contrast, Byr4 overexpression prevented Spg1 and Cdc7 localization to SPBs. These results suggest that Byr4 localization to SPBs maintains Spg1 in an inactive form, presumably by stimulating Spg1 GTPase activity with Cdc16, and that loss of Byr4 from mitotic SPBs increases the active fraction of Spg1 and thereby increases Spg1-Cdc7 binding. Byr4 localization to SPBs was decreased in spg1, cdc16, sid4, and cdc11 mutants as well as in several mutants that affect medial F-actin structures, suggesting that multiple pathways regulate Byr4 localization to SPBs.Proper cell division is essential for genome integrity. Like many eukaryotes, the fission yeast Schizosaccharomyces pombe divides by constricting an actomyosin ring that is perpendicular to the mitotic spindle (reviewed in Ref. 1). In fission yeast, a medial ring composed of F-actin and other proteins forms at the future site of cell division as cells enter mitosis. Following anaphase, this actomyosin ring constricts, a primary septum is deposited, secondary septa form on both sides of the primary septum, and the primary septum is degraded to yield two cells. Since mammals also divide by constriction of a medial actomyosin ring, fission yeast is a good model for mammalian cell division.Several fission yeast mutants perturb the temporal control of actomyosin ring constriction and septation without affecting the location or structure of the actomyosin ring (1, 2). Of these mutants, spg1, cdc7, byr4, and cdc16 are most important for this study. spg1 Ϫ and cdc7 Ϫ mutants form a medial ring, but do not constrict this ring or deposit a septum, leading to elongated, multinucleate cells (2-4). In contrast, cdc16 Ϫ and byr4 Ϫ mutants undergo repeated rounds of septation (5, 6). These mutant phenotypes suggest that Spg1 and Cdc7 are positive regulators of septation, while Byr4 and Cdc16 are negative regulators. Consistent with this notion, Spg1 or Cdc7 overexpression causes cell cycle arrest...

The ErbB-4 receptor tyrosine kinase homo-and heterodimerizes following heregulin binding, which provokes increased levels of tyrosine autophosphorylation. Unique to the ErbB family, ErbB-4 is then proteolytically cleaved by a-and c-secretase to produce an 80 kDa intracellular domain (s80 ICD) fragment. This fragment is found in both the cytoplasm and nucleus of many normal and cancer cells and can interact with transcription factors in the cytoplasm and nucleus. Since the s80 ICD lacks ectodomain sequences known to play a major role in dimerization of ErbB family members, we asked whether the s80 ICD is an active tyrosine kinase. Here, we demonstrate that the s80 ICD is a constitutively active tyrosine kinase and can form homodimers. The s80 ICD is autophosphorylated in cells and can phosphorylate an exogenous substrate in vitro. Also, the s80 ICD can coassociate and dimers are detected by chemical crosslinking. This is the first example of constitutive kinase activation and dimerization totally within the cytoplasmic domain of an ErbB receptor and suggests that the s80 ICD may function to phosphorylate substrates in the cytoplasm or nucleus.

Ectodomain cleavage of the ErbB-4 receptor tyrosine kinase generates a membrane-associated fragment of 80 kDa (m80) that has been subjected to N-terminal sequencing. The sequence obtained shows that the N terminus of this fragment begins with Ser-652 of ErbB-4. When a 12-residue peptide corresponding to ErbB-4 residues 646 -657 was incubated with recombinant tumor necrosis factor-␣-converting enzyme, fragments representing residues 646 -651 and 652-657 were obtained. These data indicate that ectodomain cleavage of ErbB-4 occurs between His-651 and Ser-652, placing the cleavage site within the ectodomain stalk region approximately 8 residues prior to the transmembrane domain. Several experiments have characterized other aspects of the m80 ErbB-4 fragment. Inhibition of ErbB-4 tyrosine kinase activity with pan-ErbB tyrosine kinase inhibitors indicates that kinase activity is stringently required for heregulindependent, but not 12-O-tetradecanoylphorbol-13-acetate-induced, ErbB-4 ectodomain cleavage and formation of the m80 fragment. When the m80 ErbB-4 fragment is generated by cell treatment with heregulin or 12-O-tetradecanoylphorbol-13-acetate, the fragment associates with intact ErbB-2. However, this fragment does not associate with the intact ErbB-4 molecule.ErbB-4 is a member of the ErbB receptor tyrosine kinase family, which also includes the epidermal growth factor receptor (ErbB-1), ErbB-2, and ErbB-3 (1). ErbB-4 and ErbB-3 bind the neuregulin (heregulin) family of growth factors, and ErbB-4 also recognizes certain growth factors in the epidermal growth factor family of ErbB-1 agonists, such as betacellulin, epiregulin, and heparin-binding epidermal growth factor. ErbB-4 heterodimerizes with ErbB-2 as do ErbB-1 and ErbB-3; however, ErbB-4 can also signal through the formation of ErbB-4 homodimers.Within the receptor tyrosine kinase family, ErbB-4 is uniquely processed (2) by a proteolytic pathway that is known to occur with certain other transmembrane proteins, such as Notch (3), the low density lipoprotein receptor-related protein (4), the amyloid precursor protein (APP) 1 (5), and the adhesion molecules CD44 (6, 7) and E-cadherin (9). The first step in this pathway involves the release of the ErbB-4 ectodomain by a cleavage that produces two fragments as follows: a 120-kDa ectodomain fragment and an 80-kDa membrane-associated fragment, designated m80 (9). The latter fragment contains the ErbB-4 transmembrane domain and the entire cytoplasmic region, including the tyrosine kinase domain. Ectodomain cleavage of ErbB-4 in cells occurs at a low constitutive or basal level (10) that can be increased by TPA (9) or by heregulin or other growth factors that bind ErbB-4 (11). Also, cleavage is potentiated by the addition to cells of pervanadate, a tyrosine phosphatase inhibitor that provokes ErbB-4 tyrosine phosphorylation (12). The ectodomain cleavage of ErbB-4 is sensitive to metalloprotease inhibitors (10) and does not occur in cells genetically deficient in tumor necrosis factor-␣-converting enzyme (TACE), a ...

No abstract

The human melanoma growth-stimulatory activities (MGSA alpha, beta, gamma/GRO) are products of immediate early genes coding for cytokines that exhibit sequence similarity to platelet factor-4 and beta-thromboglobulin. MGSA/GRO alpha has been demonstrated to partially complete for binding to the approximately 58-kDa neutrophil receptor for another beta-thromboglobulin-related chemotactic protein, IL-8. We demonstrate that when [125I]MGSA/GRO alpha was cross-linked to receptors/binding proteins from human placenta, there were two major [125I]MGSA cross-linked bands of approximately 64,000 and approximately 84,000 Mr. Because [125I]MGSA exists primarily in monomer and dimer forms at the concentrations used here, it is not clear whether the receptor/binding proteins represented by the cross-linked bands are approximately 50,000 and approximately 70,000 or approximately 58,000 and approximately 78,000 Mr. Ligand binding to the receptor proteins is associated with enhanced tyrosine phosphorylation of a number of substrates, including proteins in the same Mr range as the MGSA/GRO receptor/binding proteins.