Hypothalamic regulation of microtubule-associated protein phosphorylation in lactotrophs.
Endogenous phosphorylation of microtubule-associated proteins was examined in cultures of anterior pituitary cells from estradiol-treated rats. The cells were incubated for 1 h with 32P-orthophosphate and challenged for different times with removal of dopamine (DA), the addition of TRH in the presence of DA, or the transient removal of DA followed by addition of TRH. Microtubules were bundled by taxol followed by electrophoretic separation of the phosphorylated proteins and autoradiography. Within 10 s to 1 min of any of the treatments increased labeling of eight phosphoproteins (64, 80, 95, 110, 125, 155, 205 and 300 kDa) appeared in autoradiograms. The pattern of labeling in response to DA withdrawal was longer-lasting than that induced by TRH, whose effect disappeared by 10 min. The administration of TRH after a transient 10-min withdrawal of DA increased the magnitude and prolonged the duration of the effect of TRH. The 80-kDa microtubule-associated protein comigrated with the well characterized heat-stable, acid-soluble protein substrate for protein kinase C (PKC). The migration of the proteins following two-dimensional polyacrylamide gel electrophoresis and autoradiography was identical. Furthermore, the sequential extraction of microtubule-associated proteins followed by extraction of heat-stable, acid-soluble proteins showed a phosphoprotein of Mr 80 kDa. These observations suggest that the ubiquitous, heat-stable, acid-soluble 80-kDa phosphoprotein that is a specific substrate for PKC is associated with microtubules in lactotrophs. Furthermore, the levels of microtubule-associated phosphoprotein are increased following hormonal activation of PKC, although it is unclear whether this increase represents translocation of the phosphoprotein or phosphorylation of a previously associated protein.
Martínez de la Escalera
G
G
Department of Obstetrics, Gynecology and Reproductive Sciences, University of California School of Medicine, San Francisco.
Weiner
R I
RI
D000818:Animals; D002478:Cells, Cultured; D004298:Dopamine / Q000008:administration & dosage / Q000502:physiology; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D007031:Hypothalamus / Q000502:physiology*; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008970:Molecular Weight; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D010903:Pituitary Gland, Anterior / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013973:Thyrotropin-Releasing Hormone / Q000494:pharmacology
D008869:Microtubule-Associated Proteins; D010750:Phosphoproteins; D013973:Thyrotropin-Releasing Hormone; D004298:Dopamine