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Requirement of reversible caldesmon phosphorylation at P21-activated kinase-responsive sites for lamellipodia
Robbin D Eppinga1, Yan Li, Jenny L-C Lin
1Department of Biological Sciences, University of Iowa, Iowa City 52242-1324, USA.
Abstract:
Caldesmon is believed to be one of the key regulators for actin dynamics and thereby cell polarity, membrane extension, and cell motility. We have shown previously that stress fiber formation and cell movement are severely impaired in the cells expressing human fibroblast caldesmon fragment defective in Ca2+/CaM binding sites. Both Ser458 and Ser489, adjacent to the Ca2+/CaM-binding sites, are phosphorylated by p21-activated kinase (PAK) in vitro. Here we report that Ser458 is phosphorylated in response to cell movement. We substituted Ser458 and Ser489 on C-terminal caldesmon (CaD39) with alanine or glutamic acid to mimic under-phosphorylated (CaD39-PAKA) or constitutively phosphorylated (CaD39-PAKE) caldesmon. In vitro, CaD39-PAKE, but not CaD39-PAKA, fails to inhibit myosin ATPase activity and exhibits reduced binding to Ca2+/CaM. When stably expressed in Chinese Hamster Ovary cells, both CaD39-PAKA and CaD39-PAKE incorporate into stress fibers and localize to the leading edge of the migrating cell. Expression of CaD39-PAKE, but not CaD39-PAKA, fails to protect stress fibers from cytochalasin depolymerization. However, both mutations inhibit cell polarization and lead to defects in membrane extension and cell migration. We conclude that phosphorylation of caldesmon by PAK is a dynamic process required to regulate actin dynamics and membrane protrusions in wound-induced cell migration.
Insights
Phosphorylation of caldesmon by p21-activated kinase (PAK) is crucial for regulating actin dynamics during cell migration. This process impacts cell polarity, membrane extension, and overall cell motility.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Caldesmon regulates actin dynamics, influencing cell polarity, membrane extension, and motility.
- Previous studies showed impaired cell movement and stress fiber formation in cells with caldesmon defective in Ca2+/CaM binding.
- Serine residues 458 and 489, near Ca2+/CaM binding sites, are phosphorylated by p21-activated kinase (PAK).
Purpose of the Study:
- To investigate the role of caldesmon phosphorylation at Ser458 and Ser489 by PAK in regulating actin dynamics and cell migration.
- To determine the functional consequences of mimicking under-phosphorylated and constitutively phosphorylated caldesmon.
Main Methods:
- Site-directed mutagenesis of C-terminal caldesmon (CaD39) at Ser458 and Ser489 to alanine (CaD39-PAKA) or glutamic acid (CaD39-PAKE).
- In vitro assays to assess myosin ATPase activity and Ca2+/CaM binding.
- Stable expression of CaD39-PAKA and CaD39-PAKE in Chinese Hamster Ovary (CHO) cells.
- Analysis of stress fiber integrity, localization within migrating cells, and cell migration assays.
Main Results:
- CaD39-PAKE showed reduced Ca2+/CaM binding and failed to inhibit myosin ATPase activity in vitro.
- Both CaD39-PAKA and CaD39-PAKE localized to stress fibers and the leading edge of migrating cells.
- CaD39-PAKE expression disrupted stress fiber protection from cytochalasin, while both mutations impaired cell polarization, membrane extension, and migration.
Conclusions:
- Caldesmon phosphorylation by PAK is a dynamic process essential for regulating actin dynamics.
- This phosphorylation is critical for proper membrane protrusion and cell migration, particularly in wound-induced responses.
- Altered phosphorylation states of caldesmon significantly impact cell motility and cytoskeletal organization.
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