Microtubule-associated protein 2 (MAP2) and tau, which is involved in Alzheimer's disease, are major cytoskeletal proteins in neurons. These proteins are involved in microtubule assembly and stability. To further characterize MAP2, we took a strategy of identifying potential MAP2 binding partners. The low molecular weight MAP2c protein has 11 PXXP motifs that are conserved across species, and these PXXP motifs could be potential ligands for Src homology 3 (SH3) domains. We tested for MAP2 interaction with SH3 domain-containing proteins. All neuronal MAP2 isoforms bound specifically to the SH3 domains of c-Src and Grb2 in an in vitro glutathione S-transferase-SH3 pull-down assay. Interactions between endogenous proteins were confirmed by co-immunoprecipitation using brain lysate. All three proteins were also found co-expressed in neuronal cell bodies and dendrites. Surprisingly, the SH3 domainbinding site was mapped to the microtubule-binding domain that contains no PXXP motif. Src bound primarily the soluble, non-microtubule-associated MAP2c in vitro. This specific MAP2/SH3 domain interaction was inhibited by phosphorylation of MAP2c by the mitogenactivated protein kinase extracellular signal-regulated kinase 2 but not by protein kinase A. This phosphorylation-regulated association of MAP2 with proteins of intracellular signal transduction pathways suggests a possible link between cellular signaling and neuronal cytoskeleton, with MAP2 perhaps acting as a molecular scaffold upon which cytoskeleton-modifying proteins assemble and dissociate in response to neuronal activity.In the mammalian nervous system, the neuron-specific microtubule-associated protein MAP2 1 regulates microtubule dynamics (1) and is attributed with an important role in neurite outgrowth and dendrite development (2-6). MAP2 belongs to a family of heat stable microtubule-associated proteins found in eukaryotic cells. Other family members include MAP4, a ubiquitous protein, and tau, a neuronal protein involved in Alzheimer's disease (4, 5). Members of this family share a common microtubule-binding domain (MTBD) consisting of approximately 100 -130 amino acid residues near the carboxyl terminus of the molecule. The MTBD contains three or four tandem imperfect repeats of 18 amino acid residues separated by a variable inter-repeat region of approximately 12 amino acid residues (7).Recently, tau has been shown to bind the Src family of non-receptor tyrosine kinases (Src, Lyn, and Fyn (8)) and protein phosphatases PP1 and PP2A (9 -11). The interactions among PP1, PP2A, tau, and microtubules were postulated to regulate the phosphorylation state of tau, and alterations to these interactions could lead to the development of tauopathies seen in neurological diseases such as . This increasing body of evidence suggests that microtubule-based structural proteins like MAP2 and tau could play other physiological roles in addition to the regulation of microtubule assembly.Like tau, MAP2 is a phosphoprotein, and its cellular activities are regulated by phosphor...