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Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay
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Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

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Src-mediated tyrosine phosphorylation of PRC1 and kinastrin/SKAP on the mitotic spindle

Mariko Morii1,2, Sho Kubota1,2, Chizu Hasegawa1

  • 1Laboratory of Molecular Cell Biology, Graduate School of Pharmaceutical Sciences, Chiba University, Inohana 1-8-1, Chuo-ku, Chiba, 260-8675, Japan.

Scientific Reports
|January 29, 2021
PubMed

Insights

Src-family tyrosine kinases (SFKs) regulate mitosis by phosphorylating key spindle proteins. This phosphorylation event, particularly on Protein Regulator of Cytokinesis 1 (PRC1) and kinastrin, affects their microtubule binding during cell division.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Src-family tyrosine kinases (SFKs) are crucial for signal transduction during mitosis, including spindle formation.
  • The precise mechanisms linking SFKs to the mitotic spindle remain incompletely understood.

Purpose of the Study:

  • To elucidate the role and substrates of SFKs in mitotic spindle regulation.
  • To identify specific SFK targets and investigate their functional consequences on microtubule interactions.

Main Methods:

  • Localization studies of SFKs during mitosis, focusing on centrosome accumulation.
  • Mass spectrometry analysis of mitotic spindle preparations to identify tyrosine-phosphorylated substrates.
  • Biochemical assays using wild-type and phosphomimetic mutants of identified substrates (PRC1, kinastrin) to assess microtubule binding.

Main Results:

  • SFKs, including Fyn, were observed to accumulate in the centrosome region during mitosis in a microtubule-dependent manner.
  • Protein Regulator of Cytokinesis 1 (PRC1) and kinastrin/small kinetochore-associated protein (kinastrin/SKAP) were identified as novel SFK substrates, phosphorylated at specific tyrosine residues (Tyr-464 in PRC1, Tyr-87 in kinastrin).
  • Tyrosine phosphorylation of PRC1 and kinastrin impaired their ability to bind to microtubules, suggesting a mechanism for their delocalization.

Conclusions:

  • SFKs play a regulatory role in mitosis through the tyrosine phosphorylation of PRC1 and kinastrin.
  • This phosphorylation event is critical for the dynamic regulation of PRC1 and kinastrin association with microtubules, facilitating their proper localization during cell division.

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