Calcium/calmodulin-dependent protein kinases activity regulation

Enslen H., Tokumitsu H., Stork R J., Davis R. J., and Soderling T. R. (1996). Regulation of mitogen-activated protein kinases by a calcium/calmodulin-dependent protein kinase cascade. Proc. Natl. Acad. Sci. USA 93 10803-10808. 

Consensus sites for phosphorylation were evident in the neuronal NOS enzyme from the predicted protein sequences derived from cDNA analysis. In vitro biochemical studies indicate that nNOS can be phosphorylated by calcium/calmodulin-dependent protein kinase, cAMP-dependent protein kinase, cGMP-dependent protein kinase, and protein kinase C. Phosphorylation of nNOS by all of these enzymes decreases NOS catalytic activity in vitro (Dawson and Snyder, 1994 Bredt etal., 1992 Dinerman etal., 1994a). Calcineurin, a protein phosphatase, dephosphorylates NOS and subsequently increases its catalytic activity (T. M. Dawson etal., 1993). Multiple levels of constitutive nNOS regulation are thus possible by phosphorylation. 

The role of calcium in regulating cardiovascular physiology is presented in the fourth section of this volume. House et al. have provided an excellent review on the structure of calmodulin-dependent protein kinase II and its role in the contractility as well as proliferation and migration of VSMC. Banderali et al. have examined in detail the cellular regulation and pharmacological properties of calcium-activated potassium channels and their role in control of vascular tone by endothelium. 

In addition to the calcium-dependent activation, the activity of eNOS can also be regulated positively or negatively by the phosphorylation of the enzyme. Indeed, it has been reported that phosphorylation of the residues serine 1177 or 615 activates eNOS while the phosphorylation of the residue threonine 495, tyrosine 657, or serine 114 causes an inhibitory posttranslational modification [34]. Activation of eNOS subsequent to its phosphorylation on serine 1177 in endothelial cells has been observed in response to shear stress, bradykinin, and VEGF [34]. Several kinases are involved in the phosphorylation of serine 1177 including the protein kinase A (PKA), protein kinase B (Akt), or Ca /calmodulin-dependent protein kinases II (CaMKII) (for review, see [34]). On the other hand, eNOS is constitutively phosphorylated on threonine 495. This phosphorylation is associated in endothelial cells with a reduced binding ability of the calcium-calmoduUn complex to eNOS [35]. 

Activity is modulated by other proteins present in the membrane. These include a glycoprotein (MW 53 000) which stimulates ATPase activity 138 a 60 000 molecular weight protein, which is phosphorylated in a calmodulin-dependent fashion, affects accumulation of calcium 139 while the activity of the enzyme is affected by an endogenous kinase and phosphatase which phosphorylates and dephosphorylates the protein.140 Phospholamban is a proteolipid (MW 22 000) in cardiac SR which undergoes both cyclic AMP-dependent and calcium-calmodulin-dependent phosphorylation,141 but at different sites. All these proteins are probably involved in regulating the activity of the calcium pump. 

Fig. 11.1 Activation of MAPK pathway by Angll and ET-1 in VSMC. Stimulation of Angll and ET-1 receptors through Gq/n activation enhances the activity of PLCp. Activated PLC 3 converts PIP2 to IP3 and diacylglycerol (DAG). IP3 elevates the concentration of intracellular calcium and DAG activates PKC. PKC and/or Ca2+/calmodulin (CaM)-dependent protein kinases (CaMK) activate nonreceptor (NR) and/or receptor (R) protein tyrosine kinases. Activation of these components signals the stimulation of Ras/Raf/MEK/ERKl/2 and p70 s6k. ERK1/2 and p70 s6k are translocated to nucleus and regulate nuclear events by activating transcription factors through phosphorylation.

Phosphorylase kinase is one of the best characterized enzyme systems to illustrate the role of calcium ions in regulation of intermediary metabolism. Phosphorylase kinase is composed of four different subunits termed a (Mr 145000), /3 (MT 128000), y (A/r 45000) and 5 (Mr 17000) and has the structure (a/3y8)A [106]. Only one of its four subunits actually catalyses the phosphorylation reaction the other three subunits are regulatory and enable the enzyme complex to be activated both by calcium and cyclic AMP. The y subunit carries the catalytic activity the 8 subunit is the calcium binding protein calmodulin and is responsible for the calcium dependence of the enzyme. The a and /3 subunits are the targets for cyclic-AMP mediated regulation, both being phosphorylated by the cyclic-AMP dependent protein kinase. Calmodulin appears to interact with phosphorylase kinase in a different manner from other enzymes, since it is an integral component of the enzyme. Phosphorylase kinase has an absolute requirement for calcium, and is inactive in its absence. 

Another similarity to the catecholamine system is the fact that the initial enzyme, tryptophan hydroxylase, is subject to protein phosphorylation, which appears to increase its affinity for tetrahydrobiopterin. Of interest is the finding that, in this case, the protein kinase appears to be a specific calcium-calmodulin-dependent enzyme. While it is not known whether this regulation is physiologically significant, it has long been recognized that conditions that lead to increased activity of serotonin neurons and increased Ca " fluxes also lead to increased synthetic rates for serotonin. 

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