Evidence is presented for the differential effects of two isoforms of apolipoprotein (apo) E, apoE3 and apoE4, on neurite outgrowth and on the cytoskeleton of neuronal cells (Neuro-2a) in culture. In the presence of a lipid source, apoE3 enhances and apoE4 inhibits neurite outgrowth. Immunocytochemical studies demonstrate that there is a higher concentration of apoE3 than apoE4 in both the cell bodies and neurites. Cells treated with apoE4 showed fewer microtubules and a greatly reduced ratio of polymerized to monomeric tubulin than did cells treated with apoE3. The effect of apoE4 on depolymerization of microtubules was shown by biochemical, immunocytochemical, and ultrastructural studies. The depolymerization of microtubules and the inhibition of neurite outgrowth associated with apoE4 suggest a mechanism whereby apoE4, which has been linked to the pathogenesis of Alzheimer's disease, may prevent normal neuronal remodeling from occurring later in life, when this neurodegenerative disorder develops.

The most abundant microtubule-associated protein in sea urchin eggs and embryos is the 77 kDa echinoderm microtubule-associated protein (EMAP). EMAP localizes to the mitotic spindle as well as the interphase microtubule array and is a likely target for a cell cycle-activated kinase. To determine if EMAP is phosphorylated in vivo, sea urchin eggs and embryos were metabolically labeled with 32PO4 and a monospecific antiserum was used to immunoprecipitate EMAP from 32P-labeled eggs and embryos. In this study, we demonstrate that the 77 kDa EMAP is phosphorylated in vivo by two distinct mechanisms. In the unfertilized egg, EMAP is constitutively phosphorylated on at least five serine residues. During the first cleavage division following fertilization, EMAP is phosphorylated with a cell cycle-dependent time course. As the embryo enters mitosis, EMAP phosphorylation increases, and as the embryo exits mitosis, phosphorylation decreases. During mitosis, EMAP is phosphorylated on 10 serine residues and two-dimensional phosphopeptide mapping reveals a mitosis-specific site of phosphorylation. At all stages of the cell cycle, a 33 kDa polypeptide copurifies with the 77 kDa EMAP, regardless of phosphorylation state. Antibodies against the cdc2 kinase were used to demonstrate that the 33 kDa polypeptide is the p34cdc2 kinase. The p34cdc2 kinase copurifies with the mitotic apparatus and immunostaining indicates that the p34cdc2 kinase is concentrated at the spindle poles. Models for the interaction of the p34cdc2 kinase and the 77 kDa EMAP are presented.

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