Mechanistic Studies of cAMP-Dependent Protein Kinase Action

Bramson, H. Neal; Kaiser, E. T.; Mildvan, Albert S.; Corbin, Jackie D.

doi:10.3109/10409238409102298

Cited by 113 publications

(48 citation statements)

References 91 publications

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“…For more details see text where EAP corresponds to the non-productive dead-end complex. This mechanism compares closely with that deduced for CAMP-dependent protein kinase (see Bramson et al, 1984) for which Whitehouse and her colleagues (1983) in an elegant kinetic study have also demonstrated a steady-state ordered bi-bi mechanism. Such a mechanism, although of the rapidequilibrium type, has also recently been concluded to apply to the catalytic subunit of the protein kinase I1 (Kwiatkowski et al, 1990) which, like MLCKase, is activated by calmodulin.…”

Section: Discussionmentioning

confidence: 87%

“…From published data relating to the mechanism of action of protein kinases in general (Bramson et al, 1984) or Ca2 + /calmoddin-dependent kinases in particular, no unified mechanism has yet emerged. For example, it was reported that skeletal muscle MLCKase operates via a rapid-equilibrium random mechanism (Geuss et al, 1985) whereas the similarly Ca2 + /calmodulin-dependent catalytic domain of type I1 proCorrespondence fo A. Sobieszek, Institute of Molecular Biology, Austrian Academy of Sciences, Billorthstrasse 1 1, A-5020 Salzburg, Austria…”

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confidence: 99%

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Regulation of smooth‐muscle myosin‐light‐chain kinase

Sobieszek

1991

European Journal of Biochemistry

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The kinetic mechanism of turkey gizzard smooth muscle myosin-light-chain kinase was investigated using the isolated 20-kDa light chain of myosin as substrate. The kinetic and product inhibition patterns of the forward reaction indicated an ordered sequential mechanism in which MgATP bound first, ADP was released last. The order of substrate binding and product release was confirmed independently by competitive, dead-end inhibition patterns obtained using the non-hydrolizable ATP analog adenosine 5'-[P,y-imido]triphosphate. The mechanism was also characterized by a relatively strong product inhibition by ADP and a weak one by phosphorylated 20-kDa light-chain myosin, in addition to a significant inhibition by the latter product via a formation of a dead-end complex. [y-32P]ATP~[32P]phosphorylated light chain isotope-exchange data were consistent with the deduced mechanism and with the presence of the latter dead-end complex.Smooth muscle myosin light chain kinase (MLCKase) is a calcium-calmodulin dependent enzyme (Adelstein and Klee, 1981;Sobieszek and Barylko, 1984) that catalyses the phosphorylation of the 20-kDa light chain (Lzo) of myosin necessary for the activation of contraction in smooth muscle (for reviews see Kamm and Stull, 1985;Marston, 1982;Hartshorne and Siemankowski, 1981 ;Small and Sobieszek, 1980). In the presence of Ca2+, there is a 107-fold increase in the affinity of calmodulin for the MLCKase apoenzyme (Mamar-Bachi and Cox, 1987) that parallels the saturation of calmodulin by calcium (see Huang and King, 1985). From the demonstrated presence in the primary sequence of smooth muscle MLCKase of a stretch similar to the myosin light chain itself, a mechanism of regulation by calmodulin has recently been suggested. Thus, in the absence of calmodulin, the Lzo consensus sequence in MLCKase suppresses its own active site; with calmodulin present, conformational changes lead to opening of the complex and exposure of the active site (see Means and George, 1988).Despite advances in characterizing the domain structure, the MLCKase kinetic mechanism has still not been investigated. From published data relating to the mechanism of action of protein kinases in general (Bramson et al., 1984) or Ca2 + /calmoddin-dependent kinases in particular, no unified mechanism has yet emerged. For example, it was reported that skeletal muscle MLCKase operates via a rapid-equilibrium random mechanism (Geuss et al., 1985) whereas the similarly Ca2 + /calmodulin-dependent catalytic domain of type I1 pro- Enzyme. Myosin light chain kinase (EC 3.6.1.32).tein kinase shows a rapid-equilibrium ordered mechanism (Kwiatkowski et al., 1990). In the present study the kinetic mechanism of smooth muscle myosin light chain kinase from gizzard has been elucidated in some detail. Interestingly, although the mechanism appears to be of compulsory order type, it has some characteristics not noted previously for similar enzymes. MATERIALS AND METHODS Protein und enzyme preparationsTurkey gizzard MLCKase and calmodulin were purified ?s desc...

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Section: Discussionmentioning

confidence: 87%

mentioning

confidence: 99%

Regulation of smooth‐muscle myosin‐light‐chain kinase

Sobieszek

1991

European Journal of Biochemistry

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“…The CAMP-dependent protein kinase, which phosphorylates its physiological substrates on serine and threonine residues (for recent review cf. [21]), failed to phosphorylate any of our tyrosine-containing polymers. In contrast, several tyrosine-specific kinases (stimulated by EGF, IGF-1, and the tumor promoting phorbol ester), phosphorylated the polymers with a similar, yet distinct, specificity (Fig.…”

Section: Phosphorylation Of Tyrosine-containing Polymers By Various Pmentioning

confidence: 83%

Use of tyrosine‐containing polymers to characterize the substrate specificity of insulin and other hormone‐stimulated tyrosine kinases

Zick

Grunberger

Rees-Jones

et al. 1985

European Journal of Biochemistry

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Synthetic copolymers containing tyrosine residues were used to characterize the substrate specificity of the insulin receptor kinase and compare it to tyrosine kinases stimulated by epidermal growth factor, insulin-like growth factor-1 and phorbol ester. In partially purified receptor preparations from eight different tissues insulin best stimulated (highest V) phosphorylation of a random copolymer composed of glutamic and tyrosine residues at a 4: 1 ratio (Glu/Tyr, 4: 1). The insulin-stimulated phosphorylation of this polymer was highly significant also in receptor preparations from fresh human monocytes, where insulin binding and autophosphorylation were difficult to detect. Other tyrosine-containing polymers Ala/Glu/Lys/Tyr (6: 2: 5 : 1) and Glu/Ala/Tyr (6 : 3 : 1) were also phosphorylated by the insulin-stimulated kinase but to a lower extent. A tyrosine kinase stimulated by insulin-like growth factor-1, and one stimulated by phorbol ester also best phosphorylated the polymer Glu/Tyr (4: 1). The three kinases differed only in their capability to phosphorylate Glu/Ala/Tyr (6: 3 : 1) or Ala/Glu/Lys/ Tyr (6:2:5:1). Glu/Tyr (4:l) was a poor substrate for the epidermal growth factor receptor kinase which best phosphorylated the polymer Glu/Ala/Tyr (6 : 3 : 1). Three additional polymers: Glu/Tyr (1 : l), Glu/Ala/Tyr (1 : 1 : l), and Lys/Tyr (1 : 1) failed to serve as substrates for all four tyrosine kinases tested. Taken together these findings suggest that. (a) Hormone-sensitive tyrosine kinases have similar yet distinct substrate specificity and are likely to phosphorylate their native substrates on tyrosines adjacent to acidic (glutamic) residues. (b) Tyrosine-containing polymer substrates are highly sensitive and convenient tools to study (hormone-sensitive) tyrosine kinases whose native substrates are unknown or present at low concentrations.Phosphorylation of the insulin receptor is one of the earliest events following insulin binding [l]. The insulinstimulated receptor kinase phosphorylates itself, as well as exogenously added proteins and peptides. These include histones [2-61, casein [5, 71,, angiotensin I1 [9], and a synthetic peptide related to the site of tyrosine phosphorylation in the transforming protein kinase of Rous sarcoma virus [8, 91. While the role of receptor phosphorylation in insulin action is still unclear, the broad spectrum of exogenous substrates suggests that the insulin receptor kinase might well phosphorylate intracellular proteins to initiate the multiple effects of the hormone. Furthermore, several of the peptides and proteins phosphorylated by the insulin receptor kinase are also phosphorylated by the EGF receptor kinase [lo] and the pp60"'" kinase [ll] suggesting that these kinases might share common substrates in intact cells.In the present work, random copolymers containing tyrosine residues were used to further characterize the substrate specificity of the insulin-receptor kinase and compare it to that of other hormone-sensitive tyrosine kinases. We also desired to determi...

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“…On the other hand, there have been numerous studies describing the occurrence of cyclic nucleotidedependent protein kinases in platelets (33)(34)(35). In view of the well-established intracellular function of cAMP-dependent protein kinase (24)(25)(26), these reports focus on either intracellular or membranal functions of these kinases.…”

Section: Resultsmentioning

confidence: 99%

Platelet stimulation releases a cAMP-dependent protein kinase that specifically phosphorylates a plasma protein.

Korc‐Grodzicki

Tauber-Finkelstein

Shaltiel

1988

Proc. Natl. Acad. Sci. U.S.A.

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Rabbit serum is shown to contain a cAMPdependent protein kinase (biochemically characterized as type H) that specifically phosphorylates a 135-kDa endogenous protein. This endogenous phosphorylation can be reproduced with platelet-rich plasma, after stimulation with thrombin, but not with plasma devoid of platelets. Stimulation of isolated platelets ("washed" by gel rftration) with either thrombin or ADP brings about a release of this kinase. The supernatant of these stimulated platelets, which contains the kinase, does not undergo a cAMP-dependent endogenous phosphorylation because it does not contain the 135-kDa protein substrate. On the other hand, plasma devoid of platelets does not contain cAMP-dependent protein kinase. By combining the supernatant of the physiologically stimulated platelets with the plasma devoid of platelets, it is possible to reconstitute the system and to reproduce the specific endogenous phosphorylation of the 135-kDa target substrate. On the basis of the above evidence it is proposed that upon physiological stimulation of platelets, they release into the blood a cAMP-dependent protein kinase in addition to the well-known release of MgATP. This kinase specifically phosphorylates the 135-kDa plasma protein.Following the discovery of cAMP (1) and of cAMPdependent protein kinase (2), it became apparent that cAMPmediated protein phosphorylation plays a key role in the regulation of intracellular metabolism (3). cAMP-dependent protein kinase was shown to act as a major sensor of changes in cAMP levels within the cell, changes that take place in response to extracellular hormonal stimuli. This kinase is thus involved in the intracellular implementation of some extracellular (hormonal) messages (4), making cAMP a "second messenger" (1).cAMP-dependent protein kinases have been isolated from quite a few mammalian tissues (5). They were shown to be composed of catalytic (C) and regulatory (R) subunits assembled, in the inactive form, into an R2C2 complex (6-9). When there is a rise in the intracellular level of cAMP, this complex is activated by binding of the cyclic nucleotide to its regulatory subunits and subsequent dissociation to release free, catalytically active C subunits (6-10).The occurrence of a cAMP-triggered proteolysis of cAMPdependent protein kinase in brush-border membranes (11) led us to the discovery ofa membranal proteinase that selectively clips the C subunits of cAMP-dependent protein kinase. Further characterization of this proteinase showed (12-15) that it possesses a rather unusual biochemical specificity for C, which would suggest that C itself could be a physiological substrate ofthe kinase-splitting membranal proteinase. However, a recent study on the orientation of the proteinase showed that in cell membranes its active site faces predominantly the cell exterior (16), whereas cAMP-dependent protein kinase is so far known to act within the cell. This prompted us to consider a possible extracellular function(s) for this kinase and thus its possible occurrence in...

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Mechanistic Studies of cAMP-Dependent Protein Kinase Action

Cited by 113 publications

References 91 publications

Regulation of smooth‐muscle myosin‐light‐chain kinase

Regulation of smooth‐muscle myosin‐light‐chain kinase

Use of tyrosine‐containing polymers to characterize the substrate specificity of insulin and other hormone‐stimulated tyrosine kinases

Platelet stimulation releases a cAMP-dependent protein kinase that specifically phosphorylates a plasma protein.

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