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Regulation of the phosphorylation of human pharyngeal cell proteins by group A streptococcal surface dehydrogenase: signal transduction between streptococci and pharyngeal cells.

Pancholi V, Fischetti VA - J. Exp. Med. (1997)

Bottom Line: Intact streptococci and purified SDH induce a similar protein phosphorylation pattern with the de novo tyrosine phosphorylation of a 17-kD protein found in the membrane/particulate fraction of the pharyngeal cells.Treatment of pharyngeal cells with protein kinase inhibitors such as genistein and staurosporine significantly inhibited streptococcal invasion of pharyngeal cells.To identify the membrane receptor that elicits these signaling events, we found that SDH bound specifically to 30- and 32-kD membrane proteins in a direct ligand-binding assay.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Bacterial Pathogenesis and Immunology, The Rockefeller University, New York 10021, USA. panchov@rockvax.rockefeller.edu

ABSTRACT
Whether cell-to-cell communication results when group A streptococci interact with their target cells is unknown. Here, we report that upon contact with cultured human pharyngeal cells, both whole streptococci and purified streptococcal surface dehydrogenase (SDH) activate pharyngeal cell protein tyrosine kinase as well as protein kinase C, thus regulating the phosphorylation of cellular proteins. SDH, a major surface protein of group A streptococci, has both glyceraldehyde-3-phosphate dehydrogenase and ADP-ribosylating enzyme activities that may relate to early stages of streptococcal infection. Intact streptococci and purified SDH induce a similar protein phosphorylation pattern with the de novo tyrosine phosphorylation of a 17-kD protein found in the membrane/particulate fraction of the pharyngeal cells. However, this phosphorylation required the presence of cytosolic components. NH2-terminal amino acid sequence analysis identified the 17-kD protein as nuclear core histone H3. Both phosphotyrosine and phosphoserine-specific monoclonal antibodies reacted with the 17-kD protein by Western blot, suggesting that the binding of SDH to these pharyngeal cells elicits a novel signaling pathway that ultimately leads to activation of histone H3-specific kinases. Genistein-inhibitable phosphorylation of histone H3 indicates that tyrosine kinase plays a key role in this event. Treatment of pharyngeal cells with protein kinase inhibitors such as genistein and staurosporine significantly inhibited streptococcal invasion of pharyngeal cells. Therefore, these data indicated that streptococci/SDH-mediated phosphorylation plays a critical role in bacterial entry into the host cell. To identify the membrane receptor that elicits these signaling events, we found that SDH bound specifically to 30- and 32-kD membrane proteins in a direct ligand-binding assay. These findings clearly suggest that SDH plays an important role in cellular communication between streptococci and pharyngeal cells that may be important in host cell gene transcription, and hence in the pathogenesis of streptococcal infection.

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125I-SDH binding to  Detroit and FaDu pharyngeal  cell membrane proteins. (A) Autoradiograph showing binding  of 125I-labeled SDH to pharyngeal membrane proteins (Mem).  The membrane of Detroit and  FaDu cells were separated as described in Materials and Methods. 50 μg of total proteins was  resolved on 11% SDS-PAGE,  Western blotted on a PVDF membrane, probed with 125I-SDH (1.5 ×  105 cpm/ml, sp act: 2 × 106 cpm/μg of SDH), and autoradiographed. A  duplicate gel was similarly processed and stained with Coomassie blue. In  the membrane fraction of Detroit cells, the major labeled proteins are at  30 and 32 kD (arrows), and that in FaDu cells is a 32-kD protein (arrow). (B)  Autoradiograph showing a dose-dependent inhibition of binding of 125I-labeled SDH to the 30/32 kD Detroit pharyngeal membrane proteins in  the presence of increasing amounts (5–80 molar excess) of unlabeled purified SDH. PVDF membrane strips containing equal amounts of pharyngeal membrane proteins (100 μg/lane) were probed individually as described in A in the presence of increasing amounts of unlabeled SDH in a  final volume of 3 ml. The individual strips were then washed, dried, realigned, and autoradiographed for comparison.
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Figure 6: 125I-SDH binding to Detroit and FaDu pharyngeal cell membrane proteins. (A) Autoradiograph showing binding of 125I-labeled SDH to pharyngeal membrane proteins (Mem). The membrane of Detroit and FaDu cells were separated as described in Materials and Methods. 50 μg of total proteins was resolved on 11% SDS-PAGE, Western blotted on a PVDF membrane, probed with 125I-SDH (1.5 × 105 cpm/ml, sp act: 2 × 106 cpm/μg of SDH), and autoradiographed. A duplicate gel was similarly processed and stained with Coomassie blue. In the membrane fraction of Detroit cells, the major labeled proteins are at 30 and 32 kD (arrows), and that in FaDu cells is a 32-kD protein (arrow). (B) Autoradiograph showing a dose-dependent inhibition of binding of 125I-labeled SDH to the 30/32 kD Detroit pharyngeal membrane proteins in the presence of increasing amounts (5–80 molar excess) of unlabeled purified SDH. PVDF membrane strips containing equal amounts of pharyngeal membrane proteins (100 μg/lane) were probed individually as described in A in the presence of increasing amounts of unlabeled SDH in a final volume of 3 ml. The individual strips were then washed, dried, realigned, and autoradiographed for comparison.

Mentions: The above findings clearly indicated that the interaction of SDH/streptococci with intact pharyngeal cell surfaces results in coordinated phosphorylation of both cytosolic and membrane proteins, which in turn leads to phosphorylation of histone H3 located in the cell nucleus. Therefore, we attempted to identify the SDH-specific receptor on the surface of the pharyngeal cells. To accomplish this goal, plasma membranes were fractionated from Detroit and FaDu cells, and the proteins contained therein were probed with [125I]SDH by a direct ligand binding assay on Western blots. The results revealed that the [125I]SDH bound to Detroit pharyngeal cell membrane proteins at 30 and 32 kD (Fig. 6 A). Similarly, only a 32-kD protein of FaDu pharyngeal cell membranes bound to [125I]SDH (Fig. 6 A). Binding of 125I-labeled SDH to the 30/32-kD Detroit pharyngeal cell membrane proteins was significantly inhibited by a 40–80 molar excess of unlabeled SDH, suggesting that these proteins may serve as receptor(s) for the SDH molecule (Fig. 6 B).


Regulation of the phosphorylation of human pharyngeal cell proteins by group A streptococcal surface dehydrogenase: signal transduction between streptococci and pharyngeal cells.

Pancholi V, Fischetti VA - J. Exp. Med. (1997)

125I-SDH binding to  Detroit and FaDu pharyngeal  cell membrane proteins. (A) Autoradiograph showing binding  of 125I-labeled SDH to pharyngeal membrane proteins (Mem).  The membrane of Detroit and  FaDu cells were separated as described in Materials and Methods. 50 μg of total proteins was  resolved on 11% SDS-PAGE,  Western blotted on a PVDF membrane, probed with 125I-SDH (1.5 ×  105 cpm/ml, sp act: 2 × 106 cpm/μg of SDH), and autoradiographed. A  duplicate gel was similarly processed and stained with Coomassie blue. In  the membrane fraction of Detroit cells, the major labeled proteins are at  30 and 32 kD (arrows), and that in FaDu cells is a 32-kD protein (arrow). (B)  Autoradiograph showing a dose-dependent inhibition of binding of 125I-labeled SDH to the 30/32 kD Detroit pharyngeal membrane proteins in  the presence of increasing amounts (5–80 molar excess) of unlabeled purified SDH. PVDF membrane strips containing equal amounts of pharyngeal membrane proteins (100 μg/lane) were probed individually as described in A in the presence of increasing amounts of unlabeled SDH in a  final volume of 3 ml. The individual strips were then washed, dried, realigned, and autoradiographed for comparison.
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Figure 6: 125I-SDH binding to Detroit and FaDu pharyngeal cell membrane proteins. (A) Autoradiograph showing binding of 125I-labeled SDH to pharyngeal membrane proteins (Mem). The membrane of Detroit and FaDu cells were separated as described in Materials and Methods. 50 μg of total proteins was resolved on 11% SDS-PAGE, Western blotted on a PVDF membrane, probed with 125I-SDH (1.5 × 105 cpm/ml, sp act: 2 × 106 cpm/μg of SDH), and autoradiographed. A duplicate gel was similarly processed and stained with Coomassie blue. In the membrane fraction of Detroit cells, the major labeled proteins are at 30 and 32 kD (arrows), and that in FaDu cells is a 32-kD protein (arrow). (B) Autoradiograph showing a dose-dependent inhibition of binding of 125I-labeled SDH to the 30/32 kD Detroit pharyngeal membrane proteins in the presence of increasing amounts (5–80 molar excess) of unlabeled purified SDH. PVDF membrane strips containing equal amounts of pharyngeal membrane proteins (100 μg/lane) were probed individually as described in A in the presence of increasing amounts of unlabeled SDH in a final volume of 3 ml. The individual strips were then washed, dried, realigned, and autoradiographed for comparison.
Mentions: The above findings clearly indicated that the interaction of SDH/streptococci with intact pharyngeal cell surfaces results in coordinated phosphorylation of both cytosolic and membrane proteins, which in turn leads to phosphorylation of histone H3 located in the cell nucleus. Therefore, we attempted to identify the SDH-specific receptor on the surface of the pharyngeal cells. To accomplish this goal, plasma membranes were fractionated from Detroit and FaDu cells, and the proteins contained therein were probed with [125I]SDH by a direct ligand binding assay on Western blots. The results revealed that the [125I]SDH bound to Detroit pharyngeal cell membrane proteins at 30 and 32 kD (Fig. 6 A). Similarly, only a 32-kD protein of FaDu pharyngeal cell membranes bound to [125I]SDH (Fig. 6 A). Binding of 125I-labeled SDH to the 30/32-kD Detroit pharyngeal cell membrane proteins was significantly inhibited by a 40–80 molar excess of unlabeled SDH, suggesting that these proteins may serve as receptor(s) for the SDH molecule (Fig. 6 B).

Bottom Line: Intact streptococci and purified SDH induce a similar protein phosphorylation pattern with the de novo tyrosine phosphorylation of a 17-kD protein found in the membrane/particulate fraction of the pharyngeal cells.Treatment of pharyngeal cells with protein kinase inhibitors such as genistein and staurosporine significantly inhibited streptococcal invasion of pharyngeal cells.To identify the membrane receptor that elicits these signaling events, we found that SDH bound specifically to 30- and 32-kD membrane proteins in a direct ligand-binding assay.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Bacterial Pathogenesis and Immunology, The Rockefeller University, New York 10021, USA. panchov@rockvax.rockefeller.edu

ABSTRACT
Whether cell-to-cell communication results when group A streptococci interact with their target cells is unknown. Here, we report that upon contact with cultured human pharyngeal cells, both whole streptococci and purified streptococcal surface dehydrogenase (SDH) activate pharyngeal cell protein tyrosine kinase as well as protein kinase C, thus regulating the phosphorylation of cellular proteins. SDH, a major surface protein of group A streptococci, has both glyceraldehyde-3-phosphate dehydrogenase and ADP-ribosylating enzyme activities that may relate to early stages of streptococcal infection. Intact streptococci and purified SDH induce a similar protein phosphorylation pattern with the de novo tyrosine phosphorylation of a 17-kD protein found in the membrane/particulate fraction of the pharyngeal cells. However, this phosphorylation required the presence of cytosolic components. NH2-terminal amino acid sequence analysis identified the 17-kD protein as nuclear core histone H3. Both phosphotyrosine and phosphoserine-specific monoclonal antibodies reacted with the 17-kD protein by Western blot, suggesting that the binding of SDH to these pharyngeal cells elicits a novel signaling pathway that ultimately leads to activation of histone H3-specific kinases. Genistein-inhibitable phosphorylation of histone H3 indicates that tyrosine kinase plays a key role in this event. Treatment of pharyngeal cells with protein kinase inhibitors such as genistein and staurosporine significantly inhibited streptococcal invasion of pharyngeal cells. Therefore, these data indicated that streptococci/SDH-mediated phosphorylation plays a critical role in bacterial entry into the host cell. To identify the membrane receptor that elicits these signaling events, we found that SDH bound specifically to 30- and 32-kD membrane proteins in a direct ligand-binding assay. These findings clearly suggest that SDH plays an important role in cellular communication between streptococci and pharyngeal cells that may be important in host cell gene transcription, and hence in the pathogenesis of streptococcal infection.

Show MeSH
Related in: MedlinePlus