Autophosphorylation of carboxy-terminal residues inhibits the activity of protein kinase CK1alpha.

CK1 constitutes a protein kinase subfamily that is involved in many important physiological processes. However, there is limited knowledge about mechanisms that regulate their activity. Isoforms CK1delta and CK1epsilon were previously shown to autophosphorylate carboxy-terminal sites, a process which effectively inhibits their catalytic activity. Mass spectrometry of CK1alpha and splice variant CK1alphaL has identified the autophosphorylation of the last four carboxyl-end serines and threonines and also for CK1alphaS, the same four residues plus threonine-327 and serine-332 of the S insert. Autophosphorylation occurs while the recombinant proteins are expressed in Escherichia coli. Mutation of four carboxy-terminal phosphorylation sites of CK1alpha to alanine demonstrates that these residues are the principal but not unique sites of autophosphorylation. Treatment of autophosphorylated CK1alpha and CK1alphaS with lambda phosphatase causes an activation of 80-100% and 300%, respectively. Similar treatment fails to stimulate the CK1alpha mutants lacking autophosphorylation sites. Incubation of dephosphorylated enzymes with ATP to allow renewed autophosphorylation causes significant inhibition of CK1alpha and CK1alphaS. The substrate for these studies was a synthetic canonical peptide for CK1 (RRKDLHDDEEDEAMS*ITA). The stimulation of activity seen upon dephosphorylation of CK1alpha and CK1alphaS was also observed using the known CK1 protein substrates DARPP-32, beta-catenin, and CK2beta, which have different CK1 recognition sequences. Autophosphorylation effects on CK1alpha activity are not due to changes in Km(app) for ATP or for peptide substrate but rather to the catalytic efficiency per pmol of enzyme. This work demonstrates that CK1alpha and its splice variants can be regulated by their autophosphorylation status.

Casein kinase 2-dependent serine phosphorylation of MuSK regulates acetylcholine receptor aggregation at the neuromuscular junction.

The release of Agrin by motoneurons activates the muscle-specific receptor tyrosine kinase (MuSK) as the main organizer of subsynaptic specializations at the neuromuscular junction. MuSK downstream signaling is largely undefined. Here we show that protein kinase CK2 interacts and colocalizes with MuSK at post-synaptic specializations. We observed CK2-mediated phosphorylation of serine residues within the kinase insert (KI) of MuSK. Inhibition or knockdown of CK2, or exchange of phosphorylatable serines by alanines within the KI of MuSK, impaired acetylcholine receptor (AChR) clustering, whereas their substitution by residues that imitate constitutive phosphorylation led to aggregation of AChRs even in the presence of CK2 inhibitors. Impairment of AChR cluster formation after replacement of MuSK KI with KIs of other receptor tyrosine kinases correlates with potential CK2-dependent serine phosphorylation within KIs. MuSK activity was unchanged but AChR stability decreased in the presence of CK2 inhibitors. Muscle-specific CK2beta knockout mice develop a myasthenic phenotype due to impaired muscle endplate structure and function. This is the first description of a regulatory cross-talk between MuSK and CK2 and of a role for the KI of the receptor tyrosine kinase MuSK for the development of subsynaptic specializations.

Uncoupling substrate and activation functions of rotavirus NSP5: phosphorylation of Ser-67 by casein kinase 1 is essential for hyperphosphorylation.

Rotavirus NSP5 is a nonstructural protein that localizes in viroplasms of virus-infected cells. NSP5 interacts with NSP2 and undergoes a complex posttranslational hyperphosphorylation, generating species with reduced PAGE mobility. Here we show that NSP5 operates as an autoregulator of its own phosphorylation as a consequence of two distinct activities of the protein: substrate and activator. We developed an in vivo hyperphosphorylation assay in which two NSP5 mutant constructs are cotransfected. One of them, fused to an 11-aa tag, served as substrate whereas the other was used to map NSP5 domains required for activation. The activation and substrate activity could be uncoupled, demonstrating a hyperphosphorylation process in trans between the activator and substratum. This process involved dimerization of the two components through the 18-aa C-terminal tail. Phosphorylation of Ser-67 within the SDSAS motif (amino acids 63-67) was required to trigger hyperphosphorylation by promoting the activation function. We present evidence of casein kinase 1alpha being the protein kinase responsible for this key step as well as for the consecutive ones leading to NSP5 hyperphosphorylation.

Dual effect of lysine-rich polypeptides on the activity of protein kinase CK2.

Protein kinase CK2 (casein kinase II) is normally a heterotetramer composed of catalytic (alpha, alpha') and regulatory subunits (beta). CK2 is able to phosphorylate a large number of protein substrates but the physiological mechanisms of its regulation are still unresolved. Lysine-rich peptides such as polylysine and histone H1 are known to stimulate the catalytic activity of the holoenzyme. This activation is mediated through the CK2beta regulatory subunit. In this communication, we report that the same concentrations of lysine-rich peptides or proteins that activate the holoenzyme cause strong inhibition of the phosphorylation of proteins catalyzed by the free catalytic CK2alpha subunit. The inhibitory effect of polylysine and histone H1 is observed with several protein substrates of CK2alpha (casein, adeno E1A, transcription factor II A, and CK2beta itself). With calmodulin, however, the inhibition of CK2alpha phosphorylation caused by polylysine is much lower while with a model peptide substrate of CK2 the inhibition caused by this polycation is negligible. The inhibition of CK2alpha by polylysine is observed only at limiting concentrations of the target substrate proteins. The dual effect of polylysine and of histone H1, which results in the inhibition of CK2alpha and stimulation of the CK2 alpha(2)beta(2) tetrameric holoenzyme, has the consequence that the addition of the CK2beta, in the presence of polylysine and low concentrations of substrate protein, can cause a 242-fold stimulation of the activity of CK2alpha. Other polycationic compounds such as polyarginine and spermine do not inhibit the phosphorylation of casein by CK2alpha, indicating that the effect is specific for lysine-rich peptides. Since there is evidence that there may be free CK2alpha subunits in the nuclei of cells, where there is abundant histone H1, the inhibition of CK2alpha by this lysine-rich protein may have physiological relevance.

Role of the carboxyl terminus on the catalytic activity of protein kinase CK2alpha subunit.

Protein kinase CK2 (also known as casein kinase 2) has catalytic (alpha, alpha') and regulatory (beta) subunits. The role of carboxyl amino acids in positions from 324 to 328 was studied for Xenopus laevis CK2alpha. Deletions and mutations of these residues were produced in recombinant CK2alpha, which was assayed for kinase activity. Activity dropped 7000-fold upon deletion of amino acids 324-328. The key residues are isoleucine 327 and phenylalanine 324. A three dimensional model of CK2alpha indicates that these hydrophobic residues of helix alphaN may interact with hydrophobic residues in helix alphaE which is linked to the catalytic center.