ROCK1 interacts with JIP-3, and UVB irradiation enhances this interaction. (A) Tandem affinity purification (TAP) of ROCK1. Cellular extracts from 293ET cells transiently transfected with FLAG-HA-tagged ROCK1, with or without UVB (30 mJ/cm²) irradiation, were sequentially immunoprecipitated with anti-FLAG and anti-HA antibody-conjugated affinity resins (lanes 2, 3, 5, and 6). The ROCK1-associated polypeptides were visualized by silver staining. Mock-transfected 293ET cells were used as a control (lanes 1 and 4). Selective data from mass spectrometry analysis are shown on the right. The right panel shows detection of JIP-3 in the TAP-purified samples by Western blotting. (B) Coimmunoprecipitation of JIP-3 by ROCK1. His-tagged ROCK1 and FLAG-tagged JIP-3 were transfected in 293ET cells. Immunoprecipitation (IP) and Western blotting (WB) were performed 36 hours after transfection as indicated. (C) Endogenous association between ROCK1 and JIP-3 in U2OS cells and SCC28 cells was analyzed by coimmunoprecipitation experiments and Western blots were performed for the indicated proteins. Cells were irradiated with UVB (30 mJ/cm²) for the indicated time intervals.

ROCK1-mediated phosphorylation of JIP-3 regulates the recruitment of JNK to JIP-3 upon UVB-induced stress. (A) An increase in ROCK1-mediated phosphorylation of MYPT1 in response to UVB irradiation. Normal human primary keratinocytes, 293ET cells, and SCC28 cells were irradiated with UVB for the indicated time points. Cell lysates were subjected to immunoprecipitation with an anti-ROCK1 antibody. The immunoprecipitated pellets were assayed for ROCK1-mediated phosphorylation of the substrate recombinant MYPT1. [³²P]MYPT1 was autoradiographed, and total MYPT1 and precipitated ROCK1 proteins were stained with Coomassie blue. (B) Phosphorylation of JIP-3 by ROCK1. Purified FLAG-tagged JIP-3 (lane 2) and the control FLAG bead eluate (lane 1) were analyzed by SDS-PAGE and visualized by Coomassie blue staining (left panel). Ten micrograms of the JIP-3 protein was incubated with recombinant ROCK1 in the presence of 10 μCi [γ-³²P]ATP, with or without Y-27632 (10 μM). Phosphorylated Ser residues in JIP-3 were identified by MALDI-TOF and tandem mass spectrometry; an asterisk denotes the phosphorylated residues. (C) Effect of ROCK inhibitor on the association between JNK and JIP-3. SCC28 cells were pretreated with a ROCK inhibitor, Y-27632 (10 μM), and then exposed to UVB irradiation (30 mJ/cm²). JNK was immunoprecipitated and complexes were analyzed by Western blotting with antibodies against JIP-3 and total JNK. (D) Inhibition of the kinase activity of ROCK1 prevents UVB-induced phosphorylation of JIP-3. FLAG-JIP-3-transfected SCC28 cells were treated with either dimethyl sulfoxide (DMSO) or Y-27632 (10 μM) and were exposed to UVB irradiation for 3 hours. Total Ser-phosphorylated proteins were immunoprecipitated with an antiphosphoserine antibody. Precipitated immunocomplexes (and input proteins) were analyzed by Western blotting with an anti-FLAG antibody. (E) Mutations of Ser residues in the phosphorylation sites to Ala residues inhibited the interaction between JIP-3 and JNK that occurred upon UVB irradiation. The JIP-3 mutant constructs used for this study included WT-JIP-3, JIP-3 Mutant 1(Ser³¹⁸→Ala), and JIP-3 Mutant 2 (Ser³⁶⁸→Ala and Ser³⁶⁹→Ala).

ROCK1 acts as an activator of the pathway downstream of JNK in response to UVB-induced stress. (A) Depletion of ROCK1 abrogates the activation of the UVB-induced JNK pathway. U2OS and 293ET cells were transfected with either scrambled shRNA or ROCK1-specific shRNA followed by UVB irradiation (30 mJ/cm²). HaCaT cells were transfected with scrambled or ROCK1-specific siRNA and were then irradiated by UVB (30 mJ/cm²). Transfected cell extracts were analyzed by Western blotting with the indicated antibodies. (B) Effect of the ROCK1 inhibitor, Y-27632, on UVB-induced JNK pathway activation in normal human primary keratinocytes and U2OS cells. (C) Constitutively active ROCK1 increased the abundance of phosphorylated JNK and its target, phosphorylated c-Jun. Adenoviruses (Ad) or expression vectors expressing WT-ROCK1 or CA-ROCK1 (constitutively active) were infected or transfected into U2OS, HACAT, and 293ET cells. Cell extracts were analyzed by Western blotting with antibodies against ROCK1, phospho-γH2AX, phospho-MLC, phosphoJNK, phospho-JUN, and β-actin.

Effect of ROCK1 on the intrinsic cell death pathway. (A)Caspase-3, caspase-8, and caspase-9 activity assays were performed in cell lysates from control or ROCK1 knockdown HaCaT cells after 24 hours of UVB treatment. The results for the caspase assays are presented as the mean ± SEM (n = 3). (B) Control and ROCK1 knockdown HaCaT cells were irradiated with UVB at 30 mJ/cm², harvested at 0 and 24 hours, and whole lysates were fractionated. Fractions were assessed for the presence of cytochrome c, oxidative complex 1 (mitochondrial fraction), and IκBα (cytosolic fraction) by Western blotting analysis.

Inhibition of ROCK activity in the epidermal skin of WT mice compromises UVB-induced phosphorylation of JNK and γ-H2AX phosphorylation and apoptosis. (A) An ELISA-based assay of ROCK activity was performed on skin extracts from UVB-irradiated mice. Results are presented as the mean ± SEM (n = 3) (left panel). ROCK activity was also determined by assessing the abundance of phospho-MBS, a ROCK1 target, with or without treatment by the ROCK inhibitor HA1077 in extracts from UVB-irradiated epidermal skin (right panel). (B) HA1077 repressed UVB-induced activation of JNK and γ-H2AX in epidermal skin. WT and HA1077-treated mouse skin extracts were analyzed by Western blotting with antibodies against phospho-γ-H2AX, β-actin, phospho-JNK, and ROCK1. TUNEL and kinase assays were performed on the skin after UVB exposure (80 mJ/cm²) for 24 hours.

ROCK1 activation is essential for UVB-mediated apoptosis and JNK activation in skin in vivo. (A) UVB-mediated phosphorylation of JNK is ROCK1 dependent in vivo. Western blot analysis of the abundance of ROCK1 and phosphorylated JNK and phospho-γ-H2AX with or without UVB irradiation in the skin of WT and ROCK1^+/- mice. ROCK1^+/- mice and their WT littermates were irradiated with UVB at 160 mJ/cm². After 12 hours, mouse skins were harvested and cell lysates were analyzed by Western blotting with antibodies against ROCK1, phospho-γ-H2AX, phospho-JNK, and β-actin. (B) ROCK1^+/- mice have compromised phosphorylation of JNK in response to UVB-induced stress in the skin. Representative TUNEL staining of skin from ROCK1^+/- and WT littermates, analyzed 12 hours after the mice received 160 mJ/cm² of UVB irradiation. Immunofluorescence staining of phosphorylated JNK and phosphorylated γ-H2AX was performed on the epidermal skin from ROCK1^+/- and WT mice, with or without UVB irradiation.

Model showing the activation of the JNK pathway upon challenge by UVB. Upon UVB irradiation, ROCK1 is activated and recruits and phosphorylates JIP-3. Phosphorylated JIP-3 (P* denotes phosphorylation) triggers the phosphorylation and activation of JNK. Activated JNK causes the release of cytochrome c from the mitochondria and stimulates the intrinsic cell death pathway. Activated JNK also phosphorylates γ-H2AX, which is essential for apoptosis to occur.