A kinetic approach for the study of protein phosphatase-catalyzed regulation of protein kinase activity.
The activities of many protein kinases are regulated by phosphorylation. The phosphorylated protein kinases thus represent an important class of substrates for protein phosphatases. However, our ability to study the phosphatase-catalyzed substrate dephosphorylation has been limited in many cases by the difficulty in preparing sufficient amount of stoichiometrically phosphorylated kinases. We have applied the kinetic theory of substrate reaction during irreversible modification of enzyme activity to the study of phosphatase-catalyzed regulation of kinase activity. As an example, we measured the effect of the hematopoietic protein-tyrosine phosphatase (HePTP) on the reaction catalyzed by the fully activated, bisphosphorylated extracellular signal-regulated protein kinase 2 (ERK2/pTpY). Because only a catalytic amount of ERK2/pTpY is required, this method alleviates the need for large quantities of phospho-ERK2. Kinetic analysis of the ERK2/pTpY-catalyzed substrate reaction in the presence of HePTP leads to the determination of the rate constants for the HePTP-catalyzed dephosphorylation of free ERK2/pTpY and ERK2/pTpY*substrate(s) complexes. The data indicate that ERK2/pTpY is a highly efficient substrate for HePTP (k(cat)/K(m) = 3.05 x 10(6) M(-1) s(-1)). The data also show that binding of ATP to ERK2/pTpY has no effect on ERK2/pTpY dephosphorylation by HePTP. In contrast, binding of an Elk-1 peptide substrate to ERK2/pTpY completely blocks the HePTP action. This result indicates that phosphorylation of Tyr185 is important for ERK2 substrate recognition and that binding of the Elk-1 peptide substrate to ERK2/pTpY blocks the accessibility of pTyr185 to HePTP for dephosphorylation. Collectively, the results establish that the kinetic theory of irreversible enzyme modification can be applied to study the phosphatase catalyzed regulation of kinase activity.
Wang
Zhi-Xin
ZX
National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, Beijing, PR China.
Zhou
Bo
B
Wang
Q May
QM
Zhang
Zhong-Yin
ZY
D000255:Adenosine Triphosphate / Q000737:chemistry; D000595:Amino Acid Sequence; D002384:Catalysis; D015536:Down-Regulation; D004789:Enzyme Activation; D006412:Hematopoietic Stem Cells / Q000201:enzymology; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D010749:Phosphoprotein Phosphatases / Q000737:chemistry* / Q000378:metabolism*; D010766:Phosphorylation; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry; D013379:Substrate Specificity
D047908:Intracellular Signaling Peptides and Proteins; D000255:Adenosine Triphosphate; D019950:Mitogen-Activated Protein Kinase 1; D010749:Phosphoprotein Phosphatases; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases