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Related Experiment Video

Updated: May 6, 2026

Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

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Published on: September 18, 2013

Ser1333 phosphorylation indicates ROCKI activation.

Hsiang-Hao Chuang, Shao-Wei Liang, Zee-Fen Chang¹

¹Institute of Biochemistry and Molecular Biology, National Yang-Ming University, No, 155, Sec, 2, Linong St, Taipei 11221, Taiwan. zfchang@ym.edu.tw.

Journal of Biomedical Science

|October 31, 2013

Summary

Researchers identified a specific phosphorylation site (Ser1333) on Rho-associated protein kinase I (ROCKI) that indicates its active status. This discovery enables the development of tools to distinguish between active ROCKI and ROCKII in biological samples.

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Oligopeptide Competition Assay for Phosphorylation Site Determination

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Published on: May 18, 2017

Area of Science:

Cell Biology
Molecular Biology
Biochemistry

Background:

Rho-associated protein kinase (ROCK) isoforms, ROCKI and ROCKII, are crucial for cytoskeletal regulation and cellular processes.
ROCKI and ROCKII functions are likely non-redundant, but differentiating their activation states in samples is challenging.
Previous work identified ROCKII's Ser1366 phosphorylation site as sensitive to inhibition.

Purpose of the Study:

To identify the activity-dependent phosphorylation site in ROCKI.
To develop reagents for detecting individual ROCK isoform activation.

Main Methods:

Investigated phosphorylation sites in ROCKI sensitive to inhibition.
Developed and validated a ROCKI pSer1333-specific antibody.
Correlated ROCKI phosphorylation with downstream effectors like myosin II light chain phosphorylation.

Main Results:

Identified Ser1333 as the ROCKI phosphorylation site sensitive to inhibition.
Developed a ROCKI pSer1333-specific antibody that does not cross-react with phosphorylated ROCKII.
Demonstrated that ROCKI S1333 phosphorylation correlates with myosin II light chain phosphorylation upon RhoA stimulation.

Conclusions:

Active ROCKI is phosphorylated at the Ser1333 residue.
Phospho-specific antibodies for ROCKI (pSer1333) and ROCKII (pSer1366) allow discrimination of their individual active states.
These antibodies provide a valuable tool for studying ROCK isoform-specific roles in cells and tissues.