Normal functioning of the nervous system requires precise regulation of dendritic shape and synaptic connectivity. Here, we report a severe impairment of dendritic structures in the cerebellum and motor cortex of c-Jun N-terminal kinase 1 (JNK1)-deficient mice. Using an unbiased screen for candidate mediators, we identify the dendrite-specific high-molecular-weight microtubule-associated protein 2 (MAP2) as a JNK substrate in the brain. We subsequently show that MAP2 is phosphorylated by JNK in intact cells and that MAP2 proline-rich domain phosphorylation is decreased in JNK1Ϫ/Ϫ brain. We developed compartment-targeted JNK inhibitors to define whether a functional relationship exists between the physiologically active, cytosolic pool of JNK and dendritic architecture. Using these, we demonstrate that cytosolic, but not nuclear, JNK determines dendritic length and arbor complexity in cultured neurons. Moreover, we confirm that MAP2-dependent process elongation is enhanced after activation of JNK. Using JNK1Ϫ/Ϫ neurons, we reveal a dominant role for JNK1 over ERK in regulating dendritic arborization, whereas ERK only regulates dendrite shape under conditions in which JNK activity is low (JNK1Ϫ/Ϫ neurons). These results reveal a novel antagonism between JNK and ERK, potentially providing a mechanism for fine-tuning the dendritic arbor. Together, these data suggest that JNK phosphorylation of MAP2 plays an important role in defining dendritic architecture in the brain.

c-Jun NH2-terminal kinases (JNKs) are essential during brain development, when they regulate morphogenic changes involving cell movement and migration. In the adult, JNK determines neuronal cytoarchitecture. To help uncover the molecular effectors for JNKs in these events, we affinity purified JNK-interacting proteins from brain. This revealed that the stathmin family microtubule-destabilizing proteins SCG10, SCLIP, RB3, and RB3′ interact tightly with JNK. Furthermore, SCG10 is also phosphorylated by JNK in vivo on sites that regulate its microtubule depolymerizing activity, serines 62 and 73. SCG10-S73 phosphorylation is significantly decreased in JNK1−/− cortex, indicating that JNK1 phosphorylates SCG10 in developing forebrain. JNK phosphorylation of SCG10 determines axodendritic length in cerebrocortical cultures, and JNK site–phosphorylated SCG10 colocalizes with active JNK in embryonic brain regions undergoing neurite elongation and migration. We demonstrate that inhibition of cytoplasmic JNK and expression of SCG10-62A/73A both inhibited fluorescent tubulin recovery after photobleaching. These data suggest that JNK1 is responsible for regulation of SCG10 depolymerizing activity and neurite elongation during brain development.

The sulfated glycosaminoglycan heparan sulfate (HS) is found ubiquitously on cell surfaces, in the extracellular matrix, and intracellularly as HS proteoglycans. Because of the structural heterogeneity of HS, tissue-derived HS preparations represent a mixture of HS chains originating from different cell types and tissue loci. Monoclonal anti-HS antibodies have been employed to detect the localization of specific HS epitopes in tissues, but limited information has been available on the saccharide structures recognized by the antibodies. We have studied the saccharide epitope structures of four anti-HS antibodies, HepSS1, JM13, JM403, and 10E4, which all recognize distinct HS species as demonstrated by different patterns of immunoreactivity upon staining of embryonic rat and adult human tissues. The epitopes recognized by JM13 and HepSS1 were found almost exclusively in basement membrane HS, whereas JM403 and 10E4 reacted also with cell-associated HS species. The binding of HepSS1, JM403, and 10E4 to HS was dependent on the GlcN N-substitution of the polysaccharide rather than O-sulfation. HepSS1 thus interacted with N-sulfated HS domains, JM403 binding was critically dependent on N-unsubstituted GlcN residues, and 10E4 bound to "mixed" HS domains containing both Nacetylated and N-sulfated disaccharide units. By contrast, JM13 binding seemed to require the presence of 2-O-sulfated glucuronic acid residues. Heparan sulfate (HS)1 proteoglycans on cell surfaces and in the extracellular matrix are implicated in developmental, regenerative, and pathological processes because of their interactions with multiple proteins (1-5). These interactions are mediated mainly via the HS components of the proteoglycans, which bind to growth factors/cytokines, matrix components, enzymes, and enzyme inhibitors and thereby regulate the tissue localization and biological activities of the proteins. Characterization of HS oligosaccharides with affinity to proteins such as antithrombin (6) and peptide growth factors (7) has led to identification of specialized protein binding HS domains with ligand-specific structural distinctions. These functional domains derive from enzymatic modification in the Golgi apparatus of the primary polymerization product of HS/heparin biosynthesis, composed of alternating glucuronic acid and Nacetylglucosamine units ((GlcUA-GlcNAc) n ). The nascent polymer is first subject to partial N-deacetylation/N-sulfation of the GlcNAc residues. The modification occurs in a regioselective fashion, giving rise to (i) consecutively N-sulfated regions, (ii) regions of alternating N-acetylated and N-sulfated disaccharide units, and (iii) domains that escape modification and remain N-acetylated (5, 8). Sometimes, the N-deacetylation/Nsulfation reaction remains partial, with N-unsubstituted GlcN units as a result (9, 10). The further modification reactions, C5 epimerization of GlcUA residues into iduronic acid (IdoUA) residues and O-sulfation, all occur in the vicinity of previously incorporated N-sulfate groups. O-Sulfation...