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Talin1 phosphorylation activates β1 integrins: a novel mechanism to promote prostate cancer bone metastasis.

Jin JK, Tien PC, Cheng CJ, Song JH, Huang C, Lin SH, Gallick GE - Oncogene (2014)

Bottom Line: In contrast, reexpression of the phosphorylation-mimicking mutant talin1(S425D) led to increased β1 integrin activation and generated biologic effects opposite to talin1(S425A) expression.Immunohistochemical staining demonstrated that talin S425 phosphorylation is significantly increased in human bone metastases when compared with normal tissues, primary tumors or lymph node metastases.Together, our study reveals Cdk5-mediated phosphorylation of talin1 leading to β1 integrin activation is a novel mechanism that increases metastatic potential of PCa cells.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Genitourinary Medical Oncology, Unit 18-4, David H. Koch Center for Applied Research of Genitourinary Cancers, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA [2] The Program in Cancer Metastasis, The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, TX, USA.

ABSTRACT
Talins are adaptor proteins that regulate focal adhesion signaling by conjugating integrins to the cytoskeleton. Talins directly bind integrins and are essential for integrin activation. We previously showed that β1 integrins are activated in metastatic prostate cancer (PCa) cells, increasing PCa metastasis to lymph nodes and bone. However, how β1 integrins are activated in PCa cells is unknown. In this study, we identified a novel mechanism of β1 integrin activation. Using knockdown experiments, we first demonstrated that talin1, but not talin2, is important in β1 integrin activation. We next showed that talin1 S425 phosphorylation, but not total talin1 expression, correlates with metastatic potential of PCa cells. Expressing a non-phosphorylatable mutant, talin1(S425A), in talin1-silenced PC3-MM2 and C4-2B4 PCa cells, decreased activation of β1 integrins, integrin-mediated adhesion, motility and increased the sensitivity of the cells to anoikis. In contrast, reexpression of the phosphorylation-mimicking mutant talin1(S425D) led to increased β1 integrin activation and generated biologic effects opposite to talin1(S425A) expression. In the highly metastatic PC3-MM2 cells, expression of a non-phosphorylatable mutant, talin1(S425A), in talin1-silenced PC3-MM2 cells, abolished their ability to colonize in the bone following intracardiac injection, while reexpression of phosphorylation-mimicking mutant talin1(S425D) restored their ability to metastasize to bone. Immunohistochemical staining demonstrated that talin S425 phosphorylation is significantly increased in human bone metastases when compared with normal tissues, primary tumors or lymph node metastases. We further showed that p35 expression, an activator of Cdk5, and Cdk5 activity were increased in metastatic tumor cells, and that Cdk5 kinase activity is responsible for talin1 phosphorylation and subsequent β1 integrin activation. Together, our study reveals Cdk5-mediated phosphorylation of talin1 leading to β1 integrin activation is a novel mechanism that increases metastatic potential of PCa cells.

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Cdk5 mediates talin1 S425 phosphorylation. (a) Immunoblotting of Cdk5 and p35 in PCa cells. (b) Cdk5 kinase activity in PCa cells was measured by ADP production. *P < 0.0005 and **P < 0.0001. (c) Immunoblotting of PC3-MM2 and C4-2B4 cell lysates in which knockdown of Cdk5 inhibited talin1 S425 phosphorylation. (d) Effect of Cdk5 activity on talin1 phosphorylation by overexpression of a dominant-negative Cdk5. (e) Flow cytometric analysis of activated and total β1 integrins in Cdk5-silenced PC3-MM2 cells (top panels). Quantitation of fluorescence intensity is shown in the bottom panels. *P < 0.0005. (f) Talin1WT and mutant-expressing cells were transfected with a sh-control vector or a sh-Cdk5 vector silencing Cdk5 expression, and (g) activated and total β1 integrins were measured by flow cytometric analysis. *P < 0.005.
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Figure 4: Cdk5 mediates talin1 S425 phosphorylation. (a) Immunoblotting of Cdk5 and p35 in PCa cells. (b) Cdk5 kinase activity in PCa cells was measured by ADP production. *P < 0.0005 and **P < 0.0001. (c) Immunoblotting of PC3-MM2 and C4-2B4 cell lysates in which knockdown of Cdk5 inhibited talin1 S425 phosphorylation. (d) Effect of Cdk5 activity on talin1 phosphorylation by overexpression of a dominant-negative Cdk5. (e) Flow cytometric analysis of activated and total β1 integrins in Cdk5-silenced PC3-MM2 cells (top panels). Quantitation of fluorescence intensity is shown in the bottom panels. *P < 0.0005. (f) Talin1WT and mutant-expressing cells were transfected with a sh-control vector or a sh-Cdk5 vector silencing Cdk5 expression, and (g) activated and total β1 integrins were measured by flow cytometric analysis. *P < 0.005.

Mentions: Next, we determined the mechanism by which talin1 S425 phosphorylation is increased in metastatic PCa cells. Previous work in neuronal cells demonstrated that talin phosphorylation on S425 is catalyzed by Cdk5.17 We therefore determined whether Cdk5 was responsible for talin1 S425 phosphorylation in PCa cells. For these studies, both Cdk5 expression and kinase activity were determined. Expression of Cdk5 by immunoblotting was similar in LNCaP, C4-2B4, PC3 and PC3-MM2 cells (Figure 4a). As p35 has been shown to be the principal activator of Cdk5,19, 20 and Cdk5 and p35 are both widely expressed in PCa,21 we examined the expression of p35 in high metastatic PC3, and PC3-MM2 cells, and the low metastatic LNCaP and C4-2B4 cells. Expression of p35 correlated with metastatic potential of these cells, suggesting that p35 is responsible for Cdk5 activation (Figure 4a). Cdk5 activity (as assessed by ADP production through ADP-Glo Kinase Assay kit as described in Materials and methods) increased in PC3 (2-fold relative to LNCaP) and PC3-MM2 cells (2.5-fold relative to LNCaP; Figure 4b). Inhibition of Cdk5 by the Cdk inhibitor, roscovitine, reduced talin1 phosphorylation in PC3-MM2 cells in a time-dependent manner (Supplementary Figure S3), suggesting that Cdk5 might be responsible for talin1 phosphorylation. To examine directly whether Cdk5 mediated talin1 phosphorylation, Cdk5 was silenced in PC3-MM2 and C4-2B4 cells. The results demonstrated talin1 phosphorylation was decreased by ~90% (Figure 4c). To examine whether Cdk5 kinase activity were required for talin1 phosphorylation, PCa cells were transiently transfected with a plasmid directing the expression of a dominant-negative Myc-Cdk5.17 Expression of this dominant-negative mutant inhibited talin1 phosphorylation in both PC3-MM2 and C4-2B4 cells (Figure 4d). These results demonstrate that Cdk5 activity, but not expression, promotes talin1 S425 phosphorylation in PCa cells. We next determined whether Cdk5 regulates β1 integrin activation. Silencing Cdk5 resulted in reduced β1 integrin activation in PC3-MM2 cells (Figure 4e), suggesting that Cdk5 is required for β1 integrin activation. Next, we silenced Cdk5 in PC3-MM2 cells in which talin1 was silenced and talin1WT or talin1S425D mutants were re-expressed (Figure 4f). As expected, silencing Cdk5 inhibited β1 integrin activation in talin1WT cells, but did not affect β1 integrin activation in phospho-mimicking talin1S425D cells (Figure 4g). These observations demonstrate that Cdk5 phosphorylates talin1 on S425, and its activity promotes β1 integrin activation.


Talin1 phosphorylation activates β1 integrins: a novel mechanism to promote prostate cancer bone metastasis.

Jin JK, Tien PC, Cheng CJ, Song JH, Huang C, Lin SH, Gallick GE - Oncogene (2014)

Cdk5 mediates talin1 S425 phosphorylation. (a) Immunoblotting of Cdk5 and p35 in PCa cells. (b) Cdk5 kinase activity in PCa cells was measured by ADP production. *P < 0.0005 and **P < 0.0001. (c) Immunoblotting of PC3-MM2 and C4-2B4 cell lysates in which knockdown of Cdk5 inhibited talin1 S425 phosphorylation. (d) Effect of Cdk5 activity on talin1 phosphorylation by overexpression of a dominant-negative Cdk5. (e) Flow cytometric analysis of activated and total β1 integrins in Cdk5-silenced PC3-MM2 cells (top panels). Quantitation of fluorescence intensity is shown in the bottom panels. *P < 0.0005. (f) Talin1WT and mutant-expressing cells were transfected with a sh-control vector or a sh-Cdk5 vector silencing Cdk5 expression, and (g) activated and total β1 integrins were measured by flow cytometric analysis. *P < 0.005.
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Figure 4: Cdk5 mediates talin1 S425 phosphorylation. (a) Immunoblotting of Cdk5 and p35 in PCa cells. (b) Cdk5 kinase activity in PCa cells was measured by ADP production. *P < 0.0005 and **P < 0.0001. (c) Immunoblotting of PC3-MM2 and C4-2B4 cell lysates in which knockdown of Cdk5 inhibited talin1 S425 phosphorylation. (d) Effect of Cdk5 activity on talin1 phosphorylation by overexpression of a dominant-negative Cdk5. (e) Flow cytometric analysis of activated and total β1 integrins in Cdk5-silenced PC3-MM2 cells (top panels). Quantitation of fluorescence intensity is shown in the bottom panels. *P < 0.0005. (f) Talin1WT and mutant-expressing cells were transfected with a sh-control vector or a sh-Cdk5 vector silencing Cdk5 expression, and (g) activated and total β1 integrins were measured by flow cytometric analysis. *P < 0.005.
Mentions: Next, we determined the mechanism by which talin1 S425 phosphorylation is increased in metastatic PCa cells. Previous work in neuronal cells demonstrated that talin phosphorylation on S425 is catalyzed by Cdk5.17 We therefore determined whether Cdk5 was responsible for talin1 S425 phosphorylation in PCa cells. For these studies, both Cdk5 expression and kinase activity were determined. Expression of Cdk5 by immunoblotting was similar in LNCaP, C4-2B4, PC3 and PC3-MM2 cells (Figure 4a). As p35 has been shown to be the principal activator of Cdk5,19, 20 and Cdk5 and p35 are both widely expressed in PCa,21 we examined the expression of p35 in high metastatic PC3, and PC3-MM2 cells, and the low metastatic LNCaP and C4-2B4 cells. Expression of p35 correlated with metastatic potential of these cells, suggesting that p35 is responsible for Cdk5 activation (Figure 4a). Cdk5 activity (as assessed by ADP production through ADP-Glo Kinase Assay kit as described in Materials and methods) increased in PC3 (2-fold relative to LNCaP) and PC3-MM2 cells (2.5-fold relative to LNCaP; Figure 4b). Inhibition of Cdk5 by the Cdk inhibitor, roscovitine, reduced talin1 phosphorylation in PC3-MM2 cells in a time-dependent manner (Supplementary Figure S3), suggesting that Cdk5 might be responsible for talin1 phosphorylation. To examine directly whether Cdk5 mediated talin1 phosphorylation, Cdk5 was silenced in PC3-MM2 and C4-2B4 cells. The results demonstrated talin1 phosphorylation was decreased by ~90% (Figure 4c). To examine whether Cdk5 kinase activity were required for talin1 phosphorylation, PCa cells were transiently transfected with a plasmid directing the expression of a dominant-negative Myc-Cdk5.17 Expression of this dominant-negative mutant inhibited talin1 phosphorylation in both PC3-MM2 and C4-2B4 cells (Figure 4d). These results demonstrate that Cdk5 activity, but not expression, promotes talin1 S425 phosphorylation in PCa cells. We next determined whether Cdk5 regulates β1 integrin activation. Silencing Cdk5 resulted in reduced β1 integrin activation in PC3-MM2 cells (Figure 4e), suggesting that Cdk5 is required for β1 integrin activation. Next, we silenced Cdk5 in PC3-MM2 cells in which talin1 was silenced and talin1WT or talin1S425D mutants were re-expressed (Figure 4f). As expected, silencing Cdk5 inhibited β1 integrin activation in talin1WT cells, but did not affect β1 integrin activation in phospho-mimicking talin1S425D cells (Figure 4g). These observations demonstrate that Cdk5 phosphorylates talin1 on S425, and its activity promotes β1 integrin activation.

Bottom Line: In contrast, reexpression of the phosphorylation-mimicking mutant talin1(S425D) led to increased β1 integrin activation and generated biologic effects opposite to talin1(S425A) expression.Immunohistochemical staining demonstrated that talin S425 phosphorylation is significantly increased in human bone metastases when compared with normal tissues, primary tumors or lymph node metastases.Together, our study reveals Cdk5-mediated phosphorylation of talin1 leading to β1 integrin activation is a novel mechanism that increases metastatic potential of PCa cells.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Genitourinary Medical Oncology, Unit 18-4, David H. Koch Center for Applied Research of Genitourinary Cancers, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA [2] The Program in Cancer Metastasis, The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, TX, USA.

ABSTRACT
Talins are adaptor proteins that regulate focal adhesion signaling by conjugating integrins to the cytoskeleton. Talins directly bind integrins and are essential for integrin activation. We previously showed that β1 integrins are activated in metastatic prostate cancer (PCa) cells, increasing PCa metastasis to lymph nodes and bone. However, how β1 integrins are activated in PCa cells is unknown. In this study, we identified a novel mechanism of β1 integrin activation. Using knockdown experiments, we first demonstrated that talin1, but not talin2, is important in β1 integrin activation. We next showed that talin1 S425 phosphorylation, but not total talin1 expression, correlates with metastatic potential of PCa cells. Expressing a non-phosphorylatable mutant, talin1(S425A), in talin1-silenced PC3-MM2 and C4-2B4 PCa cells, decreased activation of β1 integrins, integrin-mediated adhesion, motility and increased the sensitivity of the cells to anoikis. In contrast, reexpression of the phosphorylation-mimicking mutant talin1(S425D) led to increased β1 integrin activation and generated biologic effects opposite to talin1(S425A) expression. In the highly metastatic PC3-MM2 cells, expression of a non-phosphorylatable mutant, talin1(S425A), in talin1-silenced PC3-MM2 cells, abolished their ability to colonize in the bone following intracardiac injection, while reexpression of phosphorylation-mimicking mutant talin1(S425D) restored their ability to metastasize to bone. Immunohistochemical staining demonstrated that talin S425 phosphorylation is significantly increased in human bone metastases when compared with normal tissues, primary tumors or lymph node metastases. We further showed that p35 expression, an activator of Cdk5, and Cdk5 activity were increased in metastatic tumor cells, and that Cdk5 kinase activity is responsible for talin1 phosphorylation and subsequent β1 integrin activation. Together, our study reveals Cdk5-mediated phosphorylation of talin1 leading to β1 integrin activation is a novel mechanism that increases metastatic potential of PCa cells.

Show MeSH
Related in: MedlinePlus