Tyrosine Phosphorylation Modulates the Activity of TRPV4 in Response to Defined Stimuli

Gerd Walz,Barbara Müller,Urs Lewandrowski,Tomasz Wegierski,Albert Sickmann

doi:10.1074/jbc.m805357200

Abstract

Src family tyrosine kinases (SFKs) regulate the function of several transient receptor potential (TRP) family members, yet their role in the regulation of the vanilloid subfamily member 4 protein (TRPV4) remains controversial. TRPV4 is a calcium-permeable channel activated by numerous physical and chemical stimuli. Here we show that SFKs mediate tyrosine phosphorylation of TRPV4 in different cell lines. Using mass spectrometric analysis, we identified two novel phosphorylation sites in the cytosolic N- and C-terminal tails of TRPV4. Substitution of either tyrosine with phenylalanine led to a substantial reduction in the overall tyrosine phosphorylation level of TRPV4, suggesting that these two tyrosines constitute major phosphorylation sites. Both mutants efficiently localized to the plasma membrane, indicating that neither tyrosine is required for trafficking of TRPV4 in the secretory pathway. Analysis of the channel function demonstrated a crucial role of the N-terminal tyrosine residue in the activation of TRPV4 by heat, mechanical (shear) stress, hypotonic cell swelling, and phorbol 12-myristate 13-acetate, but not in the activation by synthetic ligand 4alpha-phorbol 12,13-didecanoate. Furthermore, the response of TRPV4 to phorbol 12-myristate 13-acetate was SFK-dependent. Because the SFK-mediated phosphorylation of the N-terminal tyrosine occurred before TRPV4 activation, tyrosine phosphorylation appears to sensitize rather than activate this channel. Reactive oxygen species, known to mediate inflammatory pain, strongly up-regulated TRPV4 phosphorylation in the presence of SFKs. Our findings indicate that tyrosine phosphorylation of TRPV4 represents an important modulatory mechanism, which may underlie the recently described function of TRPV4 in inflammatory hyperalgesia.

Highlights

TRPV4 was initially identified as an osm-9-related channel that is activated by hypotonic cell swelling (4 – 6)
The phosphotyrosine signal in TRPV4 purified from cells pre-treated with PP2, a specific inhibitor of Src family kinases (SFKs), was reduced in a dose-dependent manner, suggesting that Src or related kinases are largely responsible for the basal level of tyrosine phosphorylation of this protein (Fig. 1A)
TRPV4 purified from Human embryonic kidney (HEK) 293T cells expressing v-src was subjected to mass spectrometry analysis to identify tyrosine phosphorylation sites in this protein

Summary

Introduction

TRPV4 was initially identified as an osm-9-related channel that is activated by hypotonic cell swelling (4 – 6). The phosphotyrosine signal in TRPV4 purified from cells pre-treated with PP2, a specific inhibitor of SFKs, was reduced in a dose-dependent manner, suggesting that Src or related kinases are largely responsible for the basal level of tyrosine phosphorylation of this protein (Fig. 1A).

Results

Conclusion