Phosphorylation and Activation of Ca2+-Calmodulin-dependent Protein Kinase IV by Ca2+-Calmodulin-dependent Protein Kinase Ia Kinase

Abstract

Purified pig brain Ca(2+)-calmodulin (CaM)-dependent protein kinase Ia kinase (Lee, J. C., and Edelman, A. M. (1994) J. Biol. Chem. 269, 2158-2164) enhances, by up to 24-fold, the activity of recombinant CaM kinase IV in a reaction also requiring Ca(2+)-CaM and MgATP. The addition of brain extract, although capable of activating CaM kinase IV by itself, provides no further activation beyond that induced by purified CaM kinase Ia kinase, consistent with the lack of a requirement of additional components for activation. Activation is accompanied by the development of significant (38%) Ca(2+)-CaM-independent CaM kinase IV activity. In parallel fashion to its activation, CaM kinase IV is phosphorylated in a CaM kinase Ia kinase-, Ca(2+)-CaM-, and MgATP-dependent manner. Phosphorylation occurs on multiple serine and threonine residues with a Ser-P:Thr-P ratio of approximately 3:1. The identical requirements for phosphorylation and activation and a linear relationship between extent of phosphorylation of CaM kinase IV and its activation state indicate that CaM kinase IV activation is induced by its phosphorylation. Replacement of Thr-196 of CaM kinase IV with a nonphosphorylatable alanine by site-directed mutagenesis abolishes both the phosphorylation and activation of CaM kinase IV, demonstrating that Thr-196 phosphorylation is essential for activation.

Mechanism of the Generation of Autonomous Activity of Ca2+/Calmodulin-dependent Protein Kinase IV

“…These characteristic features of the recombinant CaM-KIV used in this study, including generation of autonomous activity by activation and kinetic parameters of the enzyme (K m for ATP ϭ 27 M and K m for syntide-2 ϭ 264 M, Fig. 5C), were similar to the previous results obtained using enzymes expressed in Sf9 cells (16,17,19). We also performed similar experiments using CaM-KI and the same concentration (3 g/ml) of CaM-KK as used for CaM-KIV activation (Fig.…”

“…CaM-KK-It has been shown that CaM-KIV is phosphorylated at Thr 196 by CaM-KK, resulting in induction of a high level of Ca 2ϩ /CaM-dependent activity as well as generation of Ca 2ϩ / CaM-independent activity (17)(18)(19). Because CaM-KIV undergoes autophosphorylation of multiple residues subsequent to activation (36 -38), the relationship between activation of the kinase activity and Thr 196 phosphorylation has not been precisely determined in vitro.…”

“…However, this does not account for generation of the autonomous activity. At a minimum, Thr 196 phosphorylation by CaM-KK is required for generation of Ca 2ϩ /CaM-independent activity of CaM-KIV, because a mutation of Thr 196 by Ala has been shown to abolish the generation of autonomous activity (19,23). Therefore, it has not been resolved whether the Ca 2ϩ -independent activity is a direct consequence of Thr 196 phosphorylation or whether it is the result of subsequent autophosphorylation.…”

Calcium/Calmodulin-dependent Protein Kinase IV Is Cleaved by Caspase-3 and Calpain in SH-SY5Y Human Neuroblastoma Cells Undergoing Apoptosis

“…We treated SH-SY5Y cells for various times with thapsigargin and monitored CaM-stimulated protein kinase activity in the cell homogenate using the CaM kinase-selective substrate syntide-2. This peptide has been used to measure CaMK IV activity in vitro (43,62,63). Syntide-2 is homologous to phosphorylation site 2 of glycogen synthase (64).…”

Regulation and Function of the Calcium/Calmodulin-dependent Protein Kinase IV/Protein Serine/Threonine Phosphatase 2A Signaling Complex

“…As is the case for other CaM kinases, the active site of CaMKIV is sterically blocked by an autoinhibitory domain that prevents substrate binding to the enzyme. Binding of Ca 2ϩ /CaM to this region relieves autoinhibition by repositioning the autoinhibitory domain and exposes a critical residue in the activation loop, T200, to CaMKK-mediated phosphorylation (19,20). T200 phosphorylation increases CaMKIV activity and leads to the generation of Ca 2ϩ /CaM-independent, or autonomous, activity.…”