Kinase, kinases residue

The events following the activation of Ras ultimately led to the activation of MAP kinase, followed by activation of expression of immediate-early response genes. Activation of MAP kinase requires two intermediate steps, both of which involve a phosphorylation (Figure 8.5). The immediate activator of MAP kinase is MAP-kinase-kinase (also called MAP kinase-ERK kinase, or MEK), a most unusual enzyme that phosphorylates MAP kinase on both a threonine (T) and a tyrosine (Y) residue. These are in the target-sequence seven residues (LTEYVATRWYRAPE) (Table 8.1)... 

MAP kinase signaling modules typically comprise a cassette of three protein kinases (Fig. 7.2A). For example, ERK (two isoforms in mammalian cells, ERK1/2) is phosphorylated (at Thr and Tyr residues in the activation loop of its catalytic domain by an upstream MAP kinase kinase (in this case, MEK) which is, in turn, phosphorylated and activated by a MAP kinase kinase kinase, in this instance, a member of the Raf group) (Fig. 7.2A). 

FIG. 1. Douhletime mutations. The sequence of DBT and its mutations are shown. The DBT kinase domain (residues 1—292) was deduced by comparison to CKl (Gross Anderson, 1998). The kinase sub-domains are given in roman numerals, and dbfi mutants map to the catalytic domain, dco and dco are embryonic lethal mutations, dco is a pupal lethal associated with overgrowth of imaginal discs (Zilian et al 1999). The tau mutation, which arose in the Syrian hamster, affects the residue equivalent to 178 of DBT as shown. 

There are many other protein kinases that do not show any close relationship to these subfamilies. These include protein kinases with two-fold specificity, in that they can phosphorylate Ser/Thr and also Tyr residues. An example of a protein kinase with twofold specificity is the MAP kinase kinase (see Chapter 10). 

The inhibitor is boimd in an elongated form to the substrate binding surface and forms many contact points to the protein kinase. Hydrophobic residues of the inhibitor find their binding equivalents in hydrophobic pockets of the enzyme. The Arg residues important for specificity of substrate binding are in contact with Glu residues of the... 

Sjarif DR, Hellerud C, van Amstel JK, Kleijer WJ, Sperl W, Lacombe D, Sass JO, Beemer FA, Duran M, Poll-The (2004) Glycerol kinase deficiency residual activity explained by reduced transcription and enzyme conformation. Eur J Hum Genet 12 424-432... 

Once Ras is in its active form, it binds to another oncogene product called Raf, which localizes Raf to the membrane and activates its serine/threonine specific kinase activity. Among the many phosphorylation targets of Raf is another kinase called MAP kinase kinase (also called MEK), which is activated by Raf and in turn phosphorylates cytosolic MAP kinases on threonine and tyrosine residues. Activated MAP kinases (also called ERK1 and ERK2) are serine/threonine kinases that, once activated, can localize to the nucleus where they phosphorylate transcription factors such as Fos and Jun. MAP kinases also phosphorylate and activate other kinases (such as Rsk) which then pass the signal on to additional transcription factors (e.g., SRF, serum response factor). Therefore, downstream of Ras, a series of phosphorylation reactions culminate in the activation or inactivation of a variety of transcription... 

Paul R], Doerman G, Zeugner C, Riiegg JC (1983) The dependence of unloaded shortening velocity on Ca, calmodulin, and duration of contraction in chemically skinned smooth muscle. Circ Res 53 342-351 Pearson RB, Misconi LY, Kemp BE (1986) Smooth muscle myosin kinase requires residues on the COOH-terminal site of the phosphorylation site. J Biol Chem 261 25-27... 

Activation of Ras initiates a complicated protein kinase cascade that culminates in the activation of a mitogen-activated protein kinase (MAP kinase). At its core are three protein kinases. The first, Raf, is activated by binding to Ras. Raf, which is functionally a MAP kinase kinase kinase, phosphorylates serine and threonine residues on the second kinase (known as MEK in mammals), leading to its activation. Frmctionally, MEK is a MAP kinase kinase. Finally, activated MEK phosphorylates specific serine and tyrosine residues on a MAP kinase (called Erk in mammals), which is then able to phosphorylate its downstream targets. 

Phosphorylation of the tyrosine residues on growth factor receptors and activation of Ras are both short-lived events. Phos-phoprotein phosphatases reverse the phosphorylation, and the GTP bound to Ras is rapidly hydrolyzed to GDP when a GAP (see Solved Problem 6.11) binds to Ras. Therefore, transducing these short-lived signals into longer-lived serine/threonine phosphorylations on MEK and MAP kinases allows the signal, which is initiated by binding of a growth factor to its receptor, to persist. The selection of a particular pathway is achieved at the level of the MAP kinases these are inactive unless they are phosphorylated on specific serine and tyrosine residues, and the only known substrates for MAP kinase kinases are MAP kinases. 

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. 

The procedure is computationally efficient. For example, for the catalytic subunit of the mammalian cAMP-dependent protein kinase and its inhibitor, with 370 residues and 131 titratable groups, an entire calculation requires 10 hours on an SGI 02 workstation with a 175 MHz MIPS RIOOOO processor. The bulk of the computer time is spent on the FDPB calculations. The speed of the procedure is important, because it makes it possible to collect results on many systems and with many different sets of parameters in a reasonable amount of time. Thus, improvements to the method can be made based on a broad sampling of systems. 

The last part of this account will be devoted to protein kinases and protein phosphatases and some recent results we have obtained for them. Protein kinases and phosphatases are signaling biomolecules that control the level of phosphorylation and dephosphorylation of tyrosine, serine or threonine residues in other proteins, and by this means regulate a variety of fundamental cellular processes including cell growth and proliferation, cell cycle and cytoskeletal integrity. 

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