Nerve growth factor stimulates a protein kinase in PC-12 cells that phosphorylates microtubule-associated protein-2.

T Miyasaka,M V Chao,P Sherline,A R Saltiel

doi:10.1016/s0021-9258(19)39623-1

Abstract

Some of the effects of nerve growth factor (NGF) may be mediated by changes in protein phosphorylation. We have identified a protein kinase from PC-12 cells that catalyzes the phosphorylation of pig brain microtubule-associated protein (MAP)-2 in vitro. This activity is stimulated 2-4-fold in extracts from cells treated with NGF or epidermal growth factor (EGF). The partial purification and characterization of this MAP kinase indicate that it is distinct from previously described NGF-stimulated protein kinases. The NGF-stimulated kinase activity is unaffected by direct addition to the assay of the heat-stable cAMP-dependent kinase peptide inhibitor, staurosporine, or K-252A, is slightly stimulated by heparin and is inhibited by sodium fluoride and calcium ions. Treatment of cells with NGF increases the activity of the kinase within 2 min. The activity declines after 10 min, and a second phase of activation is observed at 20-30 min. Comparison of its behavior on gel permeation and sucrose density gradients indicates a molecular mass in the range of 40,000 daltons. The kinase activity is specific for ATP as substrate with a Km of 12 microM. Although the pathway of activation of MAP kinase by NGF is unknown, the stimulation can be reversed by treatment of the enzyme with alkaline phosphatase, suggesting that activation involves phosphorylation of the kinase itself. The properties and hormone sensitivity of the PC-12 MAP kinase suggest that it is similar to the previously identified, growth factor-sensitive MAP kinase from 3T3-L1 adipocytes.

Highlights

Some of the effects of nerve growth factor (NGF) may be mediated by changes in protein phospborylation
Because of the ~nctional similarities in the cellular actions of insulin and NGF, we explored the possibility that PC-12 cells might contain a similar microtubule-associated protein (MAP) kinase activity that is sensitive to activation by NGF
A number of serine protein kinases have been reported to be activated by NGF or epidermal growth factor (EGF) in PC-12 cells, including kinase N [9], CAMP-dependent kinase [27, 28], S6 kinase (l), and protein kinase C [7,8]

Summary

RESULTS

Identification of an NGF-stimulated MAP Kinase-NGF modulates the phosphorylation state of a number of proteins in PC-12 cells, including certain microtubule associated proteins [5,6]. The addition to cells of 100 nM staurosporine completely inhibited the stimulation of the kinase by NGF (Fig. 1, lane 4, and Table II), but had no effect on the activity detected in the control cells (Table II). The addition to cells of K-252A (100 nM) and H7 (25 pM) caused a 2-fold increase in the MAP kinase activities of both NGF and EGF-treated cells, but had no effect when added alone (Fig. 1, lanes 6 and 7, and Table II). The NGF-stimulated activity was reduced to basal levels by the addition of 50 mM NaF The susceptibility of this NGF-stimulated MAP kinase to these reagents indicates that this activity is similar to that described for the insulin-sensitive enzyme derived from 3T3-Ll adipocytes [17].

MAP kinase

NGF Stimulates MAP Kinase

Fraction Number

DISCUSSION