Article

Monoclonal antibody 8A2-induced retraction appears to be mediated by protein phosphorylation in goldfish retinal ganglion cell axons.

Department of Physiology, State University, Buffalo, New York 14214.
Developmental Biology (Impact Factor: 3.64). 04/1993; 156(1):230-42. DOI: 10.1006/dbio.1993.1072
Source: PubMed

ABSTRACT We have recently demonstrated that binding by monoclonal antibody (mAb) 8A2 to regenerating retinal ganglion cell axons in goldfish explants specifically induces a sustained, actin-based retraction response that is similar in most respects to a spontaneous retraction (S.G. Finnegan, V. Lemmon, and E. Koenig, Cell Motil. Cytoskeleton, 1992). Experiments were conducted to evaluate potential signal transduction pathways that may play a role in mediating retraction, using the mAb 8A2 retraction model system. Potential roles of cAMP, elevated intracellular calcium, or calmodulin-dependent processes were probed and the results did not appear to implicate them in either the induction or the maintenance of the axon retraction response. In contrast, treatment with phorbol 12-myristate 13-acetate, but not with inactive phorbol esters, induced a retraction response, although the response was more variable and less robust than that produced by mAb 8A2. However, both forms of induction were blocked by staurosporine, a nonspecific kinase inhibitor. Okadaic acid, a potent serine/threonine phosphatase inhibitor produced a very robust retraction response, and subthreshold doses significantly potentiated the retraction response induced by mAb 8A2. Genistein inhibited the mAb 8A2-induced retraction response at concentrations selective for tyrosine kinase activity in a dose-dependent manner. These findings are consistent with the hypothesis that an augmented phosphorylation state of one or more axonal proteins, perhaps catalyzed in part by protein kinase C, produces a sustained physiological retraction. In addition, tyrosine kinase may be involved in transducing surface-mediated interactions that trigger retraction, including the binding reaction signal of mAb 8A2.

0 Bookmarks
 · 
42 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: L1-mediated axon growth involves intracellular signaling, but the precise mechanisms involved are not yet clear. We report a role for the mitogen-activated protein kinase (MAPK) cascade in L1 signaling. L1 physically associates with the MAPK cascade components Raf-1, ERK2, and the previously identified p90(rsk) in brain. In vitro, ERK2 can phosphorylate L1 at Ser(1204) and Ser(1248) of the L1 cytoplasmic domain. These two serines are conserved in the L1 family of cell adhesion molecules, also being found in neurofascin and NrCAM. The ability of ERK2 to phosphorylate L1 suggests that L1 signaling could directly regulate L1 function by phosphorylation of the L1 cytoplasmic domain. In L1-expressing 3T3 cells, L1 cross-linking can activate ERK2. Remarkably, the activated ERK localizes with endocytosed vesicular L1 rather than cell surface L1, indicating that L1 internalization and signaling are coupled. Inhibition of L1 internalization with dominant-negative dynamin prevents activation of ERK. These results show that L1-generated signals activate the MAPK cascade in a manner most likely to be important in regulating L1 intracellular trafficking.
    Journal of Biological Chemistry 12/1999; 274(53):37965-73. DOI:10.1074/jbc.274.53.37965 · 4.60 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Retraction similar to that occurring spontaneously in senescent axonal fields of goldfish regenerating ganglion cell axons is reliably induced by monoclonal antibody (mAb) 8A2. The retraction response is characterized by transformation of the growth cone into a nodular motile mass, which undergoes retrograde translocation in conjunction with the contiguous column of axoplasm, generating evacuated distal strands. The growth cone-to-motile mass transformation involves a reorganization of F-actin. In addition, the reorganization of F-actin is a necessary antecedent for retrograde bulk translocation of axoplasm. Contractile tension contributes to compaction within the motile mass, while that within the column of distal axoplasm is oriented longitudinally and appears to contribute to bulk movement. As a derivative of the growth cone, the motile mass exhibits protrusive activities and a capacity to translocate independently when microtubules are partially disrupted. Apparent compressive forces cause buckling of microtubules in the adjacent segment which appear as elbow-like protrusions. Cytochalasin D blocks mAb 8A2 induced retraction and immediately arrests retrograde translocation when it is in progress; however, neither nocodazole nor taxol blocks retraction. Phalloidin and immunofluorescence double labeling of retracted axons reveals that myosin 11, MLCK, and calmodulin co-localize with dense F-actin structures within the motile mass. These results suggest that microtubules play a subordinate, passive role, and that actomyosin interactions mediate the formation of the motile mass and the retraction response. Finally, axons grown on laminin exhibit a more robust retraction response than those grown on polylysine, implicating membrane-cytoskeletal interactions as modulating factors. © 1992 Wiley-Liss, Inc.
    Cell Motility and the Cytoskeleton 01/1992; 23(4):279-301. DOI:10.1002/cm.970230407 · 4.19 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: We have previously detected an alkali-labile and developmentally regulated antigen in rat embryonic cerebral cortex, which may be 9-O-acetylsialylated GT3 ganglioside (Hirabayashi Y, Hirota M, Suzuki Y, Matsumoto M, Obata K, Ando S (1989) Neurosci Lett 106:193-98). In this study we established a mouse monoclonal antibody, 493D4, that recognizes 9-O-acetyl GT3 ganglioside, but not non-O-acetyl gangliosides. This antibody also reacted with 9-O-acetyl GD3 to a much lesser extent. By using this antibody, we found that O-acetyl GT3 as well as O-acetyl GD3 were expressed strongly in fetal murine cerebral cortex and decreased to an undetectable level after birth. With the assistance of TLC-immunostaining using 493D4 together with Q-Sepharose column chromatography, O-acetyl gangliosides of bovine brain were purified and the structural analysis showed the presence of O-acetyl GD3, O-acetyl LD1, O-acetyl GD2 and O-acetyl GD1b in the adult brain as extremely minor components. Interestingly, the antibody 493D4 could detect O-acetyl sialoglycoproteins in rat brain tissues. One of the major immunoreactive proteins was shown to be synaptophysin, an integral membrane protein specifically present in synaptic vesicles. This monoclonal antibody was therefore useful for sensitive detection of both O-acetylated gangliosides and glycoproteins with O-acetylated sialic acids.
    Glycoconjugate Journal 10/1997; 14(7):847-857. DOI:10.1023/A:1018542105832 · 1.95 Impact Factor