Nuclear translocation of prolactin: Collaboration of tyrosine kinase and protein kinase C activation in rat Nb2 node lymphoma cells

Abstract

Recent evidence has suggested that prolactin (PRL), internalized by lactogen-dependent Nb2 lymphoma cells, is actively translocated to the nucleus where it binds to PRL receptors. Moreover, the mitogenic action of PRL in these cells has been separately linked to protein tyrosyl phosphorylation and activation of protein kinase C (PKC). Therefore, the coupling of PRL internalization and nuclear translocation to the activation of these signal transduction pathways was investigated. Results from control experiments indicated that 30% of internalized and 5% total cell-associated 125I-rat PRL could be recovered within nuclei obtained from Nb2 cells previously incubated with the radiolabel for 3 h at 37 degrees C. Furthermore, internalized PRL was found to be intact and not associated with any carrier proteins. Addition of tyrosine kinase (TK) antagonists, genistein or tyrphostin, significantly reduced cell surface binding, internalization, and nuclear translocation of 125I-rat PRL. In contrast, neither the level of cell-associated nor internalized hormone differed between cells treated with the PKC antagonists, staurosporine or calphostin C, and control cultures. Instead, PKC inhibition significantly reduced nuclear PRL translocation. The inhibitory effects of the TK and PKC antagonists on PRL internalization and nuclear translocation in intact Nb2 cells were verified by immunofluorescence microscopy in parallel experiments. In other experiments, each of the kinase inhibitors blocked PRL-stimulated Nb2 cell proliferation in a concentration-dependent manner. It is concluded that activated TK and PKC collaborate in the process of PRL internalization and translocation to the nucleus. TK activation may participate in PRL receptor binding or hormone internalization while activation of PKC appears to be required for its nuclear targeting. Since TK and PKC activation are required for lactogen-stimulated Nb2 cell proliferation, we suggest that a component of the mitogenic pathway in these cells is a direct nuclear interaction of PRL.

The intranuclear prolactin/cyclophilin B complex as a transcriptional inducer

“…Intranuclear Stat5 binds to consensus Stat5 response elements, resulting in the transactivation of numerous PRL-specific genes, of which ␤-casein is the most extensively studied (9). This Stat5 transcriptional activation can be cooperatively enhanced by the glucocorticoid receptor (GR) and C͞EBP␤ (10-12).While the above-mentioned pathways are all associated with PRL-induced signaling, activation of the PRL receptor is also associated with ligand internalization via an endosomal-like pathway across the endoplasmic reticulum (ER) and nuclear envelopes (13,14). The phenomenon of protein retrotransport was initially characterized through the study of retrotranslocated viral and bacterial proteins and peptides destined for presentation on the major histocompatibility complex (15-18).…”

“…While the above-mentioned pathways are all associated with PRL-induced signaling, activation of the PRL receptor is also associated with ligand internalization via an endosomal-like pathway across the endoplasmic reticulum (ER) and nuclear envelopes (13,14). The phenomenon of protein retrotransport was initially characterized through the study of retrotranslocated viral and bacterial proteins and peptides destined for presentation on the major histocompatibility complex (15)(16)(17)(18).…”

Assessment of the mechanism by which prolactin stimulates progesterone production by early corpora lutea of pigs

“…A number of studies suggest the involvement of the PI pathway in PRL action in a variety of tissues including cultured mammary tissues, Nb2 node lymphoma cells, hypothalamic slices and astrocytes (Rillema et al 1988, DeVito et al 1993, Doppler 1994, Rao et al 1995. Activation of this pathway initiates the hydrolysis of phosphatidylinositol 4,5-bisphosphate to inositol trisphosphate and diacylglycerol.…”

“…A number of studies performed on mammary gland cells , Banerjee & Vonderhaar 1992, Fan & Rillema 1993, Nb 2 node lymphoma cells , Rao et al 1995), astrocytes (DeVito et al 1993 and hypothalamus (DeVito et al 1991) demonstrated that PKC is an important component of signal-transduction pathways induced by PRL. PKC is a family of serine/ threonine protein kinases consisting of multiple isoforms different in their structure, substrate specificity and regulatory characteristics (Nishizuka 1992, Liu 1996.…”

“…Several intracellular pathways in different tissues have been recently postulated to transduce the signal for PRL. These include the phosphatidylinositol (PI) pathway (Rillema et al 1988, DeVito et al 1993, Doppler 1994, Rao et al 1995, which encompasses phospholipase C (PLC), calcium ions and protein kinase C (PKC), the tyrosine kinase/Ras pathway (Clevenger & Medaglia 1994, Carey & Liberti 1995, Erwin et al 1995 and the JAK ( Janus tyrosine kinase)/STAT (signal transducers and activators) pathway (Campbell et al 1994, Doppler 1994, Schindler 1995, Dajee et al 1996, Watson & Burdon 1996, Hennighausen et al 1997.…”