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Modulation of type-1 protein phosphatase by synthetic peptides corresponding to the carboxyl terminus
B L Martin1, C L Shriner, D L Brautigan
1Section of Biochemistry, Brown University, Providence, RI 02912.
Abstract:
Protein phosphatase type-1 (PP-1) has a protease resistant catalytic core Mr = 35,000 (PP-35K) and carboxyl terminal segment which affects activity with various substrates. We found that micromolar concentration of a synthetic peptide, corresponding to residues 312-326 of the PP-1 carboxyl terminus (P312-326) that is missing from PP-35K, increased the phosphatase activity of PP-35K with phosphorylase and myosin light chains as substrates by decreasing the apparent Km without a change in Vm. Purified PP-1 and PP-35K were inhibited identically by okadaic acid, but peptide P312-326 only stimulated the activity of PP-35K, not full-length PP-1. Other peptides corresponding to the carboxyl terminus of phosphatase-2A or to the amino terminus of PP-1 did not affect the activity of PP-35K. A sequence conserved in PP-1 from different species, Pro-Ile-Thr-Pro-Pro was implicated as the active region because a derivative peptide, Ala-Pro-Ile-Thr-Pro-Pro-Ala, stimulated the activity of PP-35K to the same extent as peptide P312-326 although at higher concentrations. These results indicate that the carboxyl terminus of PP-1 interacts with the catalytic core to modulate its activity, and suggest that the physiological regulation of PP-1 may involve this segment.
Insights
The carboxyl terminus of Protein Phosphatase type-1 (PP-1) modulates its catalytic core (PP-35K) activity. This segment is crucial for PP-1 regulation, impacting enzyme kinetics with specific substrates.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Protein Phosphatase type-1 (PP-1) is a crucial enzyme involved in dephosphorylation.
- PP-1 consists of a catalytic core (PP-35K) and a regulatory carboxyl terminus.
- The precise role of the carboxyl terminus in modulating PP-1 activity remains to be fully elucidated.
Purpose of the Study:
- To investigate the role of the PP-1 carboxyl terminus in regulating the activity of its catalytic core (PP-35K).
- To identify specific regions within the carboxyl terminus responsible for modulating phosphatase activity.
Main Methods:
- Enzyme kinetics assays using PP-35K and various substrates (phosphorylase, myosin light chains).
- Treatment with synthetic peptides corresponding to different regions of the PP-1 carboxyl terminus.
- Comparison of peptide effects on PP-35K versus full-length PP-1 activity.
- Inhibition studies using okadaic acid.
Main Results:
- A synthetic peptide (P312-326) from the PP-1 carboxyl terminus significantly increased PP-35K activity by decreasing apparent Km.
- Peptide P312-326 stimulated PP-35K but not full-length PP-1.
- A conserved sequence (Pro-Ile-Thr-Pro-Pro) within the carboxyl terminus was implicated as an active region, with a derivative peptide showing similar stimulatory effects.
Conclusions:
- The carboxyl terminus of PP-1 directly interacts with its catalytic core to modulate enzymatic activity.
- This interaction, particularly involving the conserved Pro-Ile-Thr-Pro-Pro sequence, is critical for regulating PP-1 function.
- The findings suggest that the carboxyl terminus plays a significant role in the physiological regulation of Protein Phosphatase type-1.