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Disruption of Src Is Associated with Phenotypes Related to Williams-Beuren Syndrome and Altered Cellular Localization of TFII-I(1,2).

Sinai L, Ivakine EA, Lam E, Deurloo M, Dida J, Zirngibl RA, Jung C, Aubin JE, Feng ZP, Yeomans J, McInnes RR, Osborne LR, Roder JC - eNeuro (2015)

Bottom Line: Src is a nonreceptor protein tyrosine kinase that is expressed widely throughout the central nervous system and is involved in diverse biological functions.Src phosphorylation regulates the movement of GTF2I protein (TFII-I) between the nucleus, where it is a transcriptional activator, and the cytoplasm, where it regulates trafficking of transient receptor potential cation channel, subfamily C, member 3 (TRPC3) subunits to the plasma membrane.Here, we demonstrate altered cellular localization of both TFII-I and TRPC3 in the Src mutants, suggesting that disruption of Src can phenocopy behavioral phenotypes observed in WBS through its regulation of TFII-I.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Medical Science, University of Toronto , Toronto, Ontario, M5S 1A8, Canada ; Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital , Toronto Ontario, M5S 3E1, Canada.

ABSTRACT
Src is a nonreceptor protein tyrosine kinase that is expressed widely throughout the central nervous system and is involved in diverse biological functions. Mice homozygous for a spontaneous mutation in Src (Src (thl/thl) ) exhibited hypersociability and hyperactivity along with impairments in visuospatial, amygdala-dependent, and motor learning as well as an increased startle response to loud tones. The phenotype of Src (thl/thl) mice showed significant overlap with Williams-Beuren syndrome (WBS), a disorder caused by the deletion of several genes, including General Transcription Factor 2-I (GTF2I). Src phosphorylation regulates the movement of GTF2I protein (TFII-I) between the nucleus, where it is a transcriptional activator, and the cytoplasm, where it regulates trafficking of transient receptor potential cation channel, subfamily C, member 3 (TRPC3) subunits to the plasma membrane. Here, we demonstrate altered cellular localization of both TFII-I and TRPC3 in the Src mutants, suggesting that disruption of Src can phenocopy behavioral phenotypes observed in WBS through its regulation of TFII-I.

No MeSH data available.


Related in: MedlinePlus

Altered cellular localization of TFII-I and TRPC3 in Srcthl/thl mouse brain tissue. Western blot analysis was carried out on fractionated cell lysates from WT and Srcthl/thl adult mouse whole brain. Representative blots are shown (n = 4 WT, n = 5 Srcthl/thl). A, The nuclear fraction was probed with TFII-I antibody, with nucleolin as a loading control. B, The membrane fraction was probed with TRPC3 antibody, with n-cadherin as a control. C, Quantification of the Western blots. Data are expressed as mean ± SEM, *p < 0.05.
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Figure 9: Altered cellular localization of TFII-I and TRPC3 in Srcthl/thl mouse brain tissue. Western blot analysis was carried out on fractionated cell lysates from WT and Srcthl/thl adult mouse whole brain. Representative blots are shown (n = 4 WT, n = 5 Srcthl/thl). A, The nuclear fraction was probed with TFII-I antibody, with nucleolin as a loading control. B, The membrane fraction was probed with TRPC3 antibody, with n-cadherin as a control. C, Quantification of the Western blots. Data are expressed as mean ± SEM, *p < 0.05.

Mentions: We performed Western blot analysis of fractionated cellular compartments to compare the distribution of TFII-I and TRPC3 within the cell. Since TFII-I has been shown to require phosphorylation by Src to translocate to the nucleus in transformed cell lines (Cheriyath et al, 2002), we quantified TFII-I within the nuclear fraction of whole brain from WT and Srcthl/thl mice. There was significantly less TFII-I localized to the nucleus in cells from the Srcthl/thl mice compared with WT mice (quantification relative to loading control, WT = 1.03 ± 0.26; Srcthl/thl = 0.44 ± 0.11, Student’s t test p < 0.05; Fig. 9A,C). TRPC3 is cycled to the plasma membrane through a process that is regulated by phosphorylated TFII-I within the cytoplasm (Caraveo et al, 2006). We found significantly more TRPC3 contained within the membrane fraction in cells from the Srcthl/thl mice compared with WT mice (quantification relative to loading control, WT = 0.69 ± 0.14; Srcthl/thl = 1.44 ± 0.16, Student’s t test p < 0.05; Fig. 9B,C).


Disruption of Src Is Associated with Phenotypes Related to Williams-Beuren Syndrome and Altered Cellular Localization of TFII-I(1,2).

Sinai L, Ivakine EA, Lam E, Deurloo M, Dida J, Zirngibl RA, Jung C, Aubin JE, Feng ZP, Yeomans J, McInnes RR, Osborne LR, Roder JC - eNeuro (2015)

Altered cellular localization of TFII-I and TRPC3 in Srcthl/thl mouse brain tissue. Western blot analysis was carried out on fractionated cell lysates from WT and Srcthl/thl adult mouse whole brain. Representative blots are shown (n = 4 WT, n = 5 Srcthl/thl). A, The nuclear fraction was probed with TFII-I antibody, with nucleolin as a loading control. B, The membrane fraction was probed with TRPC3 antibody, with n-cadherin as a control. C, Quantification of the Western blots. Data are expressed as mean ± SEM, *p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4596087&req=5

Figure 9: Altered cellular localization of TFII-I and TRPC3 in Srcthl/thl mouse brain tissue. Western blot analysis was carried out on fractionated cell lysates from WT and Srcthl/thl adult mouse whole brain. Representative blots are shown (n = 4 WT, n = 5 Srcthl/thl). A, The nuclear fraction was probed with TFII-I antibody, with nucleolin as a loading control. B, The membrane fraction was probed with TRPC3 antibody, with n-cadherin as a control. C, Quantification of the Western blots. Data are expressed as mean ± SEM, *p < 0.05.
Mentions: We performed Western blot analysis of fractionated cellular compartments to compare the distribution of TFII-I and TRPC3 within the cell. Since TFII-I has been shown to require phosphorylation by Src to translocate to the nucleus in transformed cell lines (Cheriyath et al, 2002), we quantified TFII-I within the nuclear fraction of whole brain from WT and Srcthl/thl mice. There was significantly less TFII-I localized to the nucleus in cells from the Srcthl/thl mice compared with WT mice (quantification relative to loading control, WT = 1.03 ± 0.26; Srcthl/thl = 0.44 ± 0.11, Student’s t test p < 0.05; Fig. 9A,C). TRPC3 is cycled to the plasma membrane through a process that is regulated by phosphorylated TFII-I within the cytoplasm (Caraveo et al, 2006). We found significantly more TRPC3 contained within the membrane fraction in cells from the Srcthl/thl mice compared with WT mice (quantification relative to loading control, WT = 0.69 ± 0.14; Srcthl/thl = 1.44 ± 0.16, Student’s t test p < 0.05; Fig. 9B,C).

Bottom Line: Src is a nonreceptor protein tyrosine kinase that is expressed widely throughout the central nervous system and is involved in diverse biological functions.Src phosphorylation regulates the movement of GTF2I protein (TFII-I) between the nucleus, where it is a transcriptional activator, and the cytoplasm, where it regulates trafficking of transient receptor potential cation channel, subfamily C, member 3 (TRPC3) subunits to the plasma membrane.Here, we demonstrate altered cellular localization of both TFII-I and TRPC3 in the Src mutants, suggesting that disruption of Src can phenocopy behavioral phenotypes observed in WBS through its regulation of TFII-I.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Medical Science, University of Toronto , Toronto, Ontario, M5S 1A8, Canada ; Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital , Toronto Ontario, M5S 3E1, Canada.

ABSTRACT
Src is a nonreceptor protein tyrosine kinase that is expressed widely throughout the central nervous system and is involved in diverse biological functions. Mice homozygous for a spontaneous mutation in Src (Src (thl/thl) ) exhibited hypersociability and hyperactivity along with impairments in visuospatial, amygdala-dependent, and motor learning as well as an increased startle response to loud tones. The phenotype of Src (thl/thl) mice showed significant overlap with Williams-Beuren syndrome (WBS), a disorder caused by the deletion of several genes, including General Transcription Factor 2-I (GTF2I). Src phosphorylation regulates the movement of GTF2I protein (TFII-I) between the nucleus, where it is a transcriptional activator, and the cytoplasm, where it regulates trafficking of transient receptor potential cation channel, subfamily C, member 3 (TRPC3) subunits to the plasma membrane. Here, we demonstrate altered cellular localization of both TFII-I and TRPC3 in the Src mutants, suggesting that disruption of Src can phenocopy behavioral phenotypes observed in WBS through its regulation of TFII-I.

No MeSH data available.


Related in: MedlinePlus