Abstract
The cell stress response encompasses the range of intracellular events required for adaptation to stimuli detrimental to cell survival. Although the c-Jun N-terminal kinase (JNK) is a stress-activated kinase that can promote either cell survival or death in response to detrimental stimuli, the JNK-regulated mechanisms involved in survival are not fully characterized. Here we show that in response to hyperosmotic stress, JNK phosphorylates a key cytoplasmic microtubule regulatory protein, stathmin (STMN), on conserved Ser-25 and Ser-38 residues. In in vitro biochemical studies, we identified STMN Ser-38 as the critical residue required for efficient phosphorylation by JNK and identified a novel kinase interaction domain in STMN required for recognition by JNK. We revealed that JNK was required for microtubule stabilization in response to hyperosmotic stress. Importantly, we also demonstrated a novel cytoprotective function for STMN, as the knockdown of STMN levels by siRNA was sufficient to augment viability in response to hyperosmotic stress. Our findings show that JNK targeting of STMN represents a novel stress-activated cytoprotective mechanism involving microtubule network changes.
Highlights
Mote cell survival and proliferation [4]
When we investigated the changes in the mitogen-activated protein kinase (MAPK), the phosphorylation of all three MAPK subfamilies was increased in response to hyperosmotic stress (Fig. 1B)
STMN during neurotrophic stimulation in PC12 cells. These results indicate that an important link between Jun N-terminal kinase (JNK) activation and STMN phosphorylation was not restricted to fibroblasts during their response to osmotic stress and that JNK and extracellular signalregulated kinase (ERK) make different contributions to STMN phosphorylation under conditions of stress and growth factor stimulation
Summary
Antibodies and Reagents—Antibodies: STMN, ␣tubulin (Sigma); p-JNK, p-p38, (Ser(P)-73)-c-Jun, c-Jun, and p-ERK (Cell Signaling); (Ser(P)-16)-STMN, ERK1, p38, GAPDH (Santa Cruz Biotechnology), (Ser(P)-25)-STMN, (Ser(P)-38)STMN (Abcam); JNK (BD Biosciences Australia). Media, and supplements were obtained from Invitrogen. Whereas the protein kinases targeting STMN in growth factor-stimulated signaling have been defined (e.g. ERK, PKA, and cyclin-dependent kinase), less is known about the stress-activated protein kinase(s) that drives STMN phosphorylation and inactivation. STMN Ser-16 phosphorylation showed similar kinetics (i.e. plateau at 15 min sorbitol treatment and appearing as multiple bands) (Fig. 1A). Immunoblot analysis with a pan-STMN antibody indicated that STMN levels were not markedly changed during exposure to sorbitol (Fig. 1A). JNK phosphorylation, indicative of its activation, was evident at 5 min and peaked at 1–2 h after sorbitol treatment (Fig. 1B). Our results indicate that the buffer before being denatured in Laemmli buffer
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