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Requirement for Src family protein tyrosine kinases in G2 for fibroblast cell division
S Roche1, S Fumagalli, S A Courtneidge
1Institut National de la Santé et de la Recherche Médicale (INSERM) faculté de Pharmacie, Montpellier, France.
Abstract:
The protein tyrosine kinase c-Src is transiently activated at the transition from the G2 phase to mitosis in the cell cycle of mammalian fibroblasts. Fyn and Yes, the other members of the Src family present in fibroblasts, were also found to be activated at mitosis. In cells microinjected with a neutralizing antibody specific for Src, Fyn, and Yes (anti-cst.1) during G2, cell division was inhibited by 75 percent. The block occurred before nuclear envelope breakdown. Antibodies specific for phosphatidylinositol-3 kinase alpha and phospholipase C-gamma 1 had no effect. Microinjection of the Src homology 2 (SH2) domain of Fyn was also inhibitory. Functional redundancy between members of the Src family was observed; a Src-specific antibody had no effect in NIH 3T3 cells but inhibited cell division in fibroblasts in which the only functional Src family kinase was Src itself. Thus, Src family kinases and proteins associating with their SH2 domains are required for entry into mitosis.
Insights
Src family kinases, including c-Src, Fyn, and Yes, are crucial for cell division. Inhibiting these protein tyrosine kinases during the G2 phase blocks mitosis, highlighting their essential role in cell cycle progression.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Protein tyrosine kinases (PTKs) play critical roles in regulating cellular processes.
- The Src family of kinases (SFKs) are non-receptor tyrosine kinases involved in various signaling pathways.
- Cell cycle progression is tightly regulated by a complex network of proteins and signaling events.
Purpose of the Study:
- To investigate the role of Src family kinases (SFKs) in mammalian cell cycle progression, specifically during the G2 to M phase transition.
- To determine which specific SFKs are activated during mitosis and their contribution to cell division.
- To elucidate the molecular mechanisms by which SFKs regulate entry into mitosis.
Main Methods:
- Utilized microinjection of neutralizing antibodies specific for c-Src, Fyn, and Yes into mammalian fibroblasts.
- Employed antibodies targeting phosphatidylinositol-3 kinase alpha and phospholipase C-gamma 1 as controls.
- Introduced the Src homology 2 (SH2) domain of Fyn to assess its inhibitory potential.
- Compared the effects of SFK inhibition in different cell lines, including NIH 3T3 cells.
Main Results:
- Src family kinases (c-Src, Fyn, and Yes) are transiently activated during the G2 to M phase transition.
- Microinjection of a pan-SFK neutralizing antibody (anti-cst.1) during G2 inhibited cell division by 75%, preceding nuclear envelope breakdown.
- Inhibition of phosphatidylinositol-3 kinase alpha and phospholipase C-gamma 1 did not affect cell division.
- Microinjection of the Fyn SH2 domain also inhibited cell division.
- Demonstrated functional redundancy among SFKs, with Src-specific antibodies affecting cells dependent solely on Src for SFK activity.
Conclusions:
- Src family kinases are essential regulators of the G2 to M phase transition in mammalian cell cycle.
- Proteins interacting with the SH2 domains of SFKs are critical for mitotic entry.
- SFKs and their associated proteins are required for successful progression into mitosis.