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Protein kinase C spatially and temporally regulates gap junctional communication during human wound repair via phosphorylation of connexin43 on serine368.

Richards TS, Dunn CA, Carter WG, Usui ML, Olerud JE, Lampe PD - J. Cell Biol. (2004)

Bottom Line: Phosphorylation of connexin43 (Cx43) on serine368 (S368) has been shown to decrease gap junctional communication via a reduction in unitary channel conductance.However, 24 h after wounding, but not at 6 or 72 h, pS368 levels were dramatically increased in basal keratinocytes and essentially lost from suprabasal layers adjacent to the wound (i.e., within 200 microm of it).Our evidence indicates that PKC-dependent phosphorylation of Cx43 at S368 creates dynamic communication compartments that can temporally and spatially regulate wound healing.

View Article: PubMed Central - PubMed

Affiliation: Division of Basic and Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.

ABSTRACT
Phosphorylation of connexin43 (Cx43) on serine368 (S368) has been shown to decrease gap junctional communication via a reduction in unitary channel conductance. Examination of phosphoserine368 (pS368) in normal human skin tissue using a phosphorylation site-specific antibody showed relatively even distribution throughout the epidermal layers. However, 24 h after wounding, but not at 6 or 72 h, pS368 levels were dramatically increased in basal keratinocytes and essentially lost from suprabasal layers adjacent to the wound (i.e., within 200 microm of it). Scratch wounding of primary human keratinocytes caused a protein kinase C (PKC)-dependent increase in pS368 in cells adjacent to the scratch, with a time course similar to that found in the wounds. Keratinocytes at the edge of the scratch also transferred dye much less efficiently at 24 h, in a manner dependent on PKC. However, keratinocyte migration to fill the scratch required early (within <6 h) gap junctional communication. Our evidence indicates that PKC-dependent phosphorylation of Cx43 at S368 creates dynamic communication compartments that can temporally and spatially regulate wound healing.

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pS368 levels rise relative to total Cx43 in response to scratch wounding in a manner dependent on PKC activity. (A) Time course after wounding of the ratio of pS368 to total Cx43, normalized to the zero time level. (B) pS368/Cx43 levels 24 h after wounding in the presence and absence of kinase inhibitors, normalized to the control level. Control was not scrape wounded but was harvested at the same time as the wounded samples. BIM, bisindolylmaleimide (200 nM); Ro-31, Ro-31-8220 (100 nM); and Gö6976 (100 nM). (A and B) Error bars represent standard error of the mean. (C) The COOH-terminal region of Cx43 (residues 246–382) was phosphorylated with PKCɛ and immunoblotted as described in Materials and methods. Phosphorylation and total Cx43 were detected with antibodies to pS368 and the COOH-terminal region of Cx43. (D) Representative immunoblot of the PKC inhibitor data summarized in B. Note that the migration of the pS368 signal overlays with the fastest migrating species of Cx43 and there is a higher level of total Cx43 in the control because cells were not lost during scratch wounding.
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fig3: pS368 levels rise relative to total Cx43 in response to scratch wounding in a manner dependent on PKC activity. (A) Time course after wounding of the ratio of pS368 to total Cx43, normalized to the zero time level. (B) pS368/Cx43 levels 24 h after wounding in the presence and absence of kinase inhibitors, normalized to the control level. Control was not scrape wounded but was harvested at the same time as the wounded samples. BIM, bisindolylmaleimide (200 nM); Ro-31, Ro-31-8220 (100 nM); and Gö6976 (100 nM). (A and B) Error bars represent standard error of the mean. (C) The COOH-terminal region of Cx43 (residues 246–382) was phosphorylated with PKCɛ and immunoblotted as described in Materials and methods. Phosphorylation and total Cx43 were detected with antibodies to pS368 and the COOH-terminal region of Cx43. (D) Representative immunoblot of the PKC inhibitor data summarized in B. Note that the migration of the pS368 signal overlays with the fastest migrating species of Cx43 and there is a higher level of total Cx43 in the control because cells were not lost during scratch wounding.

Mentions: Whether the level of pS368 throughout the scratch-wounded culture was generally elevated is difficult to determine by immunofluorescence, so we performed a time course experiment and immunoblotted for pS368 and total Cx43 at 3, 6, 10, 24, 48, and 72 h after the scratch wound (Fig. 3 A). The phosphorylation level at S368 increased up to 24 h and leveled at 48 h, after which proliferation and migration completely closed the scratch wound. The extent of phosphorylation then decreased, at 72 h, to control levels. Given the immunofluorescence results, we would expect that the cells near the scratch contribute most of the increase in pS368 signal.


Protein kinase C spatially and temporally regulates gap junctional communication during human wound repair via phosphorylation of connexin43 on serine368.

Richards TS, Dunn CA, Carter WG, Usui ML, Olerud JE, Lampe PD - J. Cell Biol. (2004)

pS368 levels rise relative to total Cx43 in response to scratch wounding in a manner dependent on PKC activity. (A) Time course after wounding of the ratio of pS368 to total Cx43, normalized to the zero time level. (B) pS368/Cx43 levels 24 h after wounding in the presence and absence of kinase inhibitors, normalized to the control level. Control was not scrape wounded but was harvested at the same time as the wounded samples. BIM, bisindolylmaleimide (200 nM); Ro-31, Ro-31-8220 (100 nM); and Gö6976 (100 nM). (A and B) Error bars represent standard error of the mean. (C) The COOH-terminal region of Cx43 (residues 246–382) was phosphorylated with PKCɛ and immunoblotted as described in Materials and methods. Phosphorylation and total Cx43 were detected with antibodies to pS368 and the COOH-terminal region of Cx43. (D) Representative immunoblot of the PKC inhibitor data summarized in B. Note that the migration of the pS368 signal overlays with the fastest migrating species of Cx43 and there is a higher level of total Cx43 in the control because cells were not lost during scratch wounding.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172473&req=5

fig3: pS368 levels rise relative to total Cx43 in response to scratch wounding in a manner dependent on PKC activity. (A) Time course after wounding of the ratio of pS368 to total Cx43, normalized to the zero time level. (B) pS368/Cx43 levels 24 h after wounding in the presence and absence of kinase inhibitors, normalized to the control level. Control was not scrape wounded but was harvested at the same time as the wounded samples. BIM, bisindolylmaleimide (200 nM); Ro-31, Ro-31-8220 (100 nM); and Gö6976 (100 nM). (A and B) Error bars represent standard error of the mean. (C) The COOH-terminal region of Cx43 (residues 246–382) was phosphorylated with PKCɛ and immunoblotted as described in Materials and methods. Phosphorylation and total Cx43 were detected with antibodies to pS368 and the COOH-terminal region of Cx43. (D) Representative immunoblot of the PKC inhibitor data summarized in B. Note that the migration of the pS368 signal overlays with the fastest migrating species of Cx43 and there is a higher level of total Cx43 in the control because cells were not lost during scratch wounding.
Mentions: Whether the level of pS368 throughout the scratch-wounded culture was generally elevated is difficult to determine by immunofluorescence, so we performed a time course experiment and immunoblotted for pS368 and total Cx43 at 3, 6, 10, 24, 48, and 72 h after the scratch wound (Fig. 3 A). The phosphorylation level at S368 increased up to 24 h and leveled at 48 h, after which proliferation and migration completely closed the scratch wound. The extent of phosphorylation then decreased, at 72 h, to control levels. Given the immunofluorescence results, we would expect that the cells near the scratch contribute most of the increase in pS368 signal.

Bottom Line: Phosphorylation of connexin43 (Cx43) on serine368 (S368) has been shown to decrease gap junctional communication via a reduction in unitary channel conductance.However, 24 h after wounding, but not at 6 or 72 h, pS368 levels were dramatically increased in basal keratinocytes and essentially lost from suprabasal layers adjacent to the wound (i.e., within 200 microm of it).Our evidence indicates that PKC-dependent phosphorylation of Cx43 at S368 creates dynamic communication compartments that can temporally and spatially regulate wound healing.

View Article: PubMed Central - PubMed

Affiliation: Division of Basic and Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.

ABSTRACT
Phosphorylation of connexin43 (Cx43) on serine368 (S368) has been shown to decrease gap junctional communication via a reduction in unitary channel conductance. Examination of phosphoserine368 (pS368) in normal human skin tissue using a phosphorylation site-specific antibody showed relatively even distribution throughout the epidermal layers. However, 24 h after wounding, but not at 6 or 72 h, pS368 levels were dramatically increased in basal keratinocytes and essentially lost from suprabasal layers adjacent to the wound (i.e., within 200 microm of it). Scratch wounding of primary human keratinocytes caused a protein kinase C (PKC)-dependent increase in pS368 in cells adjacent to the scratch, with a time course similar to that found in the wounds. Keratinocytes at the edge of the scratch also transferred dye much less efficiently at 24 h, in a manner dependent on PKC. However, keratinocyte migration to fill the scratch required early (within <6 h) gap junctional communication. Our evidence indicates that PKC-dependent phosphorylation of Cx43 at S368 creates dynamic communication compartments that can temporally and spatially regulate wound healing.

Show MeSH
Related in: MedlinePlus