Related Experiment Videos
Specificity of the polycation-stimulated (type-2A) and ATP,Mg-dependent (type-1) protein phosphatases toward
P Agostinis1, R Derua, S Sarno
1Afdeling Biochemie, Faculteit Geneeskunde, Katholieke Universiteit te Leuven, Belgium.
Abstract:
p34cdc2 kinase, a critical regulator of the cell cycle, has been shown to recognize the consensus sequence S/TP in proteins such as histone H1, the retinoblastoma gene product RB and the carboxyl-terminal domain of eukaryotic RNA polymerase II. Using phosphorylated synthetic peptides, representing the p34cdc2 phosphorylation sites in these proteins and histone H1 protein as substrates, we investigated the substrate specificity of the different oligomeric forms of the polycation-stimulated (PCS/type-2A) protein phosphatase and the active catalytic subunit of the ATP,Mg-dependent (AMDc/type 1) protein phosphatase. The results show that the oligomeric structure of the PCS phosphatases is an important determinant for efficient dephosphorylation. The trimeric PCSH1 and PCSM phosphatases are about 10-20-fold-better histone H1 phosphatases than the dimeric PCSH2 and PCSL phosphatases and about 100-fold better than the catalytic subunit (PCSC), suggesting a regulatory role for the 72-kDa, 65-kDa and 55-kDa subunits. The RB peptide = INGS(P)PRT(P)PRRGQNR, is preferred over phosphorylase a (8-fold) by the PCSH1 phosphatase and is about a 40-fold and 95-fold-better substrate for the PCSH1 phosphatase than for the PCSM and PCSL phosphatases, respectively. The primary structure surrounding the S/T(P)P motif, by itself a strong negative determinant for dephosphorylation, can harbour positive features which relieve the constraint imposed by the carboxyl-terminal proline. Thus, the RB peptide INGS(P)PRT(P)PRRGQNR, in which the T(P)P configuration is preferred over the S(P)P sequence, is an extremely good and specific substrate for the PCSH1 phosphatase (Km = 10 microM, Vmax = 3882 nmol.min-1.mg-1). The AMDC phosphatase is a poor phosphatase for all the phosphopeptides tested, unless Mn2+ is added. Its histone H1 phosphatase activity is much less sensitive than its phosphorylase a and phosphopeptide phosphatase activity to inhibition by the modulator or inhibitor-1. The results strongly suggest a role for the trimeric PCSH1 phosphatase in reversing the p34cdc2 phosphorylations.
Insights
The trimeric PCSH1 phosphatase efficiently reverses cell cycle regulator p34cdc2 kinase phosphorylations, unlike other forms. Oligomeric structure and specific peptide sequences are key determinants for phosphatase activity.
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Biology
Background:
- p34cdc2 kinase regulates the cell cycle by phosphorylating proteins like histone H1, RB, and RNA polymerase II at S/TP motifs.
- Protein phosphatases counteract kinase activity, but their substrate specificity and regulation are not fully understood.
Purpose of the Study:
- To investigate the substrate specificity of polycation-stimulated (PCS/type-2A) and ATP,Mg-dependent (AMDc/type 1) protein phosphatases.
- To determine the role of oligomeric structure in PCS phosphatase activity.
- To identify determinants of substrate recognition for specific phosphorylation sites.
Main Methods:
- Used phosphorylated synthetic peptides mimicking p34cdc2 phosphorylation sites and histone H1 protein as substrates.
- Assayed activity of different oligomeric forms of PCS phosphatases (trimeric PCSH1, PCSM, PCSH2, PCSL, and catalytic subunit PCSC) and AMDc phosphatase.
- Determined kinetic parameters (Km, Vmax) for specific peptide substrates.
Main Results:
- Trimeric PCSH1 and PCSM phosphatases were significantly more active against histone H1 than dimeric or monomeric forms.
- The retinoblastoma (RB) peptide (INGS(P)RTPPRRGQNR) was a highly preferred and specific substrate for PCSH1 phosphatase.
- The ATP,Mg-dependent (AMDc) phosphatase showed poor activity unless Mn2+ was added and was less sensitive to inhibitors.
Conclusions:
- Oligomeric structure is a critical determinant for PCS phosphatase substrate specificity and efficiency.
- The trimeric PCSH1 phosphatase likely plays a key role in reversing p34cdc2 kinase-mediated cell cycle phosphorylations.
- Primary sequence context surrounding phosphorylation sites significantly influences dephosphorylation by specific phosphatases.