Stimulation of Ca(2+)/calmodulin-dependent protein kinase phosphatase by polycations.
Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKPase) dephosphorylates and regulates multifunctional Ca(2+)/calmodulin-dependent protein kinases (CaMKs). One of the prominent features of CaMKPase is stimulation of phosphatase activity by polycations such as poly-L-lysine (poly(Lys)). Using various polycations, basicity and molecular weight of the polymer proved to be important for the stimulation. Surface plasmon resonance (SPR) analysis showed that CaMKIV(T196D), which mimics CaMKPase substrate, and CaMKPase could form tight complexes with poly(Lys). Pull-down binding experiments suggested that the formation of a tightly associated ternary complex consisting of CaMKPase, poly(Lys), and phosphorylated CaMKIV is essential for stimulation. Dilution experiments also supported this contention. Poly(Lys) failed to stimulate a CaMKPase mutant in which a Glu cluster corresponding to residues 101-109 in the N-terminal domain was deleted, and the mutant could not interact with poly(Lys) in the presence of Mn(2+). Thus, the Glu cluster appeared to be the binding site for polycations and to play a pivotal role in the polycation stimulation of CaMKPase activity.
Ishida
Atsuhiko
A
Department of Biochemistry, Asahikawa Medical College, Japan. aishida@asahikawa-med.ac.jp
Kameshita
Isamu
I
Kitani
Takako
T
Okuno
Sachiko
S
Takeuchi
Masayuki
M
Fujisawa
Hitoshi
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D054734:Calcium-Calmodulin-Dependent Protein Kinase Type 4; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D008345:Manganese / Q000378:metabolism / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D010749:Phosphoprotein Phosphatases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011073:Polyamines / Q000378:metabolism* / Q000494:pharmacology; D000071228:Polyelectrolytes; D011107:Polylysine / Q000494:pharmacology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020349:Surface Plasmon Resonance
D054734:Calcium-Calmodulin-Dependent Protein Kinase Type 4; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D008345:Manganese; D010749:Phosphoprotein Phosphatases; D011073:Polyamines; D000071228:Polyelectrolytes; D011107:Polylysine; D011994:Recombinant Proteins; C110531:calmodulin dependent protein kinase II phosphatase