Autophosphorylating serine/threonine kinase

Most protein serine-threonine kinases undergo autophosphorylation 399... 

Most protein serine-threonine kinases undergo autophosphorylation. The autophosphorylation of most protein kinases is associated with an increase in kinase activity [4, 10]. In some instances, such as with the RII subunit of PKA, autophosphorylation represents a positive feedback mechanism for kinase activation, in this case by enhancing the rate of dissociation of the RII and C subunits. In the case of CaMKII, autophosphorylation causes the catalytic activity of the enzyme to become independent of Ca2+ and calmodulin. This means that the enzyme, activated originally in response to elevated cellular Ca2+, remains active after Ca2+ concentrations have returned to baseline. By this mechanism, neurotransmitters that activate CaMKII can produce relatively long-lived alterations in neuronal function. In other instances, such as with the receptor-associated protein tyrosine kinases (discussed in Ch. 24), autophosphorylation is an obligatory step in the sequence of molecular events through which those kinases are activated and produce physiological effects. 

Several other Ca +ZCaM-dependent serine/ threonine kinases have been identified that can be distinguished from CaM-kinase II on the basis of native size, substrate specificity, and autophosphorylation properties. One of these is CaM-kinase Gr or IV, which is a multifunctional kinase found primarily in brain (Means et al., 1991). CaM-kinase IV has a peptide substrate specificity that is similar to that of CaM-kinase II, and it also undergoes Ca +ZCaM-dependent autophosphorylation on a serine residue (Frangakis et al.,... 

Photl is a membrane-associated 120 kD serine/threonine kinase that undergoes autophosphorylation upon absorption of near-UV and blue light. - Both phototropins thus represent light-activated serine-threonine protein kinases. The formation of the flavin-C(4a)-cysteinyl adduct upon light absorption causes a conformational change of the apoprotein that is believed to activate the kinase activity. ... 

In vitro, with CaATP as a substrate, E. coli dnaK autophosphorylates exclusively at Thr-199 (McCarty and Walker, 1991). It does not auto-phosphorylate when MgATP is used as a substrate. In vivo, dnaK is found to be phosphorylated on serine as well as on threonine residues (Rieul et al., 1987). Under normal growth conditions, phosphorylation is primarily on serine when E. coli is infected with bacteriophage M13, the phosphorylation shifts predominantly to threonine, with minor phosphorylation of serine. The specific target residues of in vivo phosphorylation of dnaK have not yet been determined. The disparity between the in vitro results (autophosphorylation exclusively at Thr-199 an absolute requirement for CaATP) and the in vivo results (phosphorylation on both serine and threonine residues, under conditions in which MgATP would be presumed to be the available intracellular substrate nucleotide) raises the questions of (1) whether the specific autophosphorylation of Thr-199 observed in vitro also occurs in vivo, or whether it may be an artifactual side reaction when the larger Ca ion is substituted for Mg" at the active site of the protein, and (2) whether there is a serine/threonine protein kinase that specifically phosphorylates dnaK in vivo. 

Heterogeneous phosphorylation is often a problem when kinases are expressed in insect cells. Multiple approaches have been used to solve this problem. Proteins have been completely dephosphorylated by incubation with A protein phosphatase or alkaline phosphatase [38, 39, 56]. Ion exchange and isoelectric focusing chromatography have been used to separate proteins with multiple phosphorylation states. An y-aminophenyl ATP-sepharose column was used to separate different phosphory-lated states of human c-Src [34]. Alternatively, serine/threonine or tyrosine phosphorylation sites can be mutated to alanine or phenylalanine, respectively [42]. For tyrosine kinases with multiple autophosphorylation sites, the active site aspartic acid can be mutated to an asparagine, creating a kinase dead mutant [57]. 

Fig. 6.5 Signalling through SMAD proteins. Receptor complexes, formed with transforming growth factor.p (TGF-p) and related factors, activins, and bone morphogenetic proteins (BMPs), consist of two serine/threonine receptor kinases, the type II and type I receptors. Binding of the ligand results In autophosphorylation of type I receptors. The activated type I receptors in turn phosphoryiate and activate R-SMADs SMADs 1,...

Barrett CB, Erikson E, Mailer JL. 1992 A purified S6 kinase kinase from Xenopus eggs activates S6 kinase II and autophosphorylates on serine, threonine, and tyrosine residues. J Biol Chem 267(7) 4408-4415. 

In comparison to the level of cellular serine or threonine phosphorylation, protein tyrosine phosphorylation occurs at quite low levels in normal cells but dramatically increases upon oncogenic transformation or stimulation. Since the first discovery in 1978 that the transforming protein from Rous sarcoma virus (pp60vsrc) exhibited intrinsic kinase activity/5 protein kinase activity has also been shown to be inherent to other growth factor receptors such as epidermal growth factor receptor and the insulin receptor,[6 91 and to involve autophosphorylation processes. The diverse biochemical activity exhibited by protein tyrosine phosphorylation has stimulated the development of chemical methods for the preparation of phosphorylated peptides for use as substrates in elucidating the biochemical and physiological activity of phosphorylated site(s). 

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