Calcium activation of ERK mediated by calmodulin kinase I.

Gary A. Wayman,Thomas R. Soderling,Naohito Nozaki,John M. Schmitt

doi:10.1074/jbc.m401501200

Abstract

Elevated intracellular Ca(2+) triggers numerous signaling pathways including protein kinases such as the calmodulin-dependent kinases (CaMKs) and the extracellular signal-regulated kinases (ERKs). In the present study we examined Ca(2+)-dependent "cross-talk" between these two protein kinase families. Using a combination of pharmacological inhibitors and dominant-negative kinases (dnKinase), we identified a requirement for CaMKK acting through CaMKI in the stimulation of ERKs upon depolarization of the neuroblastoma cell line, NG108. Depolarization stimulated prolonged ERK and JNK activation that was blocked by the CaMKK inhibitor, STO-609; this inhibition of ERK activation by STO-609 was rescued by expression of a STO-609-insensitive mutant of CaMKK. However, activation of ERK by epidermal growth factor or carbachol were not suppressed by inhibition of CaMKK, indicating specificity for this "cross-talk." To identify the downstream target of CaMKK that mediated ERK activation upon depolarization, dnKinases were expressed. The dnCaMKI completely suppressed ERK2 activation whereas dnAKT/PKB or nuclear-targeted dnCaMKIV, other substrates for CaMKK, were not inhibitory. ERK activation upon depolarization or transfection with constitutively active (ca) CaMKI was blocked by dnRas. Additionally, depolarization of NG108 cells promoted neurite outgrowth, and this effect was blocked by inhibition of either CaMKK (STO-609) or ERK (UO126). Co-transfection with caCaMKK plus caCaMKI also stimulated neurite outgrowth that was blocked by inhibition of ERK (UO126). These data are the first to suggest that ERK activation and neurite outgrowth in response to depolarization are mediated by CaMKK activation of CaMKI.

Highlights

One of the most ubiquitous cellular signaling mechanisms is the extracellular signal-regulated kinase (ERK)1 pathway
Depolarization of NG108 cells with KCl resulted in robust ERK activation that was evident within 1 min (Fig. 2A) and maintained at both 20 and 60 min (Fig. 1A)
We have focused on the sustained activation of ERK (20 and 60 min.) rather than its rapid stimulation (1–5 min) because sustained activation appears to be important for physiological phenomena such as gene transcription and neurite outgrowth [5, 8, 48, 49]

Summary

Calcium Activation of ERK Mediated by Calmodulin Kinase I*

The response of the ERK pathway to various stimuli can be cell type-specific and/or -dependent on regulation of different subcellular pools of small G proteins [17, 18, 21]. Some of these pathways are mediated in part through elevation of intracellular calcium, which can play an important role in ERK activation, especially in neurons. We have carefully evaluated the roles of CaMKs in ERK activation using more specific molecular and pharmacological inhibitors to determine the physiologic significance of this pathway in neurite outgrowth upon cellular depolarization

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION