Proline-directed protein kinases,

The proline-directed protein kinases (PDPK), which phosphorylate tau on serine or threonine residues that are followed by a proline residue. This group includes tau protein kinase II (cdk5), cdk2, MAP kinase (p38), INK, and other SAPKs (Baumann et al., 1993 Holzer et al., 1994 Goedert et al., 1997). 

In this method, phosphotransferase activity is used to measure MAPK activity in extracts of smooth muscle tissue. MAPK-specific activity is detected using a synthetic peptide as a substrate in the phosphotransferase reaction. The use of myelin basic protein will result in the detection of all kinases that phos-phorylate this substrate (including MAPK, protein kinase C, and other protein kinases whereas the use of a synthetic peptide, designed as a proline-directed protein kinase substrate, results in the detection of MAPK-specific activity, exclusively (Adam et al., 1995 Clark-Lewis et ah, 1991). 

MAPK is more likely to be the physiologically relevant "caldesmon kinase" since there is no evidence to date for p34 i 2 or any PDPK other than MAPK, in arterial muscle (Adam and Hathaway, 1993 Adamef a/., 1995). With the use of a specific peptide substrate, the 42- and 44-kDa isoforms of MAPK (p42mapk and p44MAPK) were found to contain all the proline-directed protein kinase activity in contractile smooth muscle. However, it is possible that PDPKs other than MAPK exist in smooth muscle and that (1) the activity of these are not detected using this peptide substrate and (2) they phosphorylate caldesmon. 

Fig. 7. Possible two-step regulation of protein function by proline-directed protein kinases and Pinl (a hypothesis) [63-65]. Phosphorylation (P) of some proteins on Ser/Thr-Pro sites by proline-directed kinases, such as cdc2 and MAP kinases, creates binding sites for Pinl. Pinl prefers to recognize the phosphorylated Ser/Thr-Pro with a hydrophobic amino acid (hy) stretch at the amino terminus, and it isomerizes the peptide bond adjacent to proline in the phosphorylated Ser/Thr-Pro sequence. This action of Pinl changes the shape of the whole protein from conformation 1 to conformation 2, which might have the ability to interact with other proteins, to translocate to other cellular compartments, or to change its life span. This regulation may be critical for the coordinated G,/M progression in mitosis.

The nonproline-directed protein kinases (NPDPKs) such as tau-tubulin kinase 1 and 2, protein kinase A (PKA), protein kinase C (PKC), PKB/AKT, calmodulin (CaM) kinase II, MARK kinases, or CK I and n that modify residues close to acidic residues mainly in exons 2 and 3. NPDPKs modify Ser or Thr residues that are not followed by prolines (Kitano-Takahashi et al., 2007 Sergeant et al.,... 

In addition to allosteric regulation of PKC activity by membrane phospholipids, it has become apparent that phosphorylation of the kinase itself is an important regulatory mechanism. Using site-directed mutagenesis approaches, phosphorylation of PKCa on Thr 97 (Cazaubon etal., 1994) or PKC(3II on Thr (Orr and Newton, 1994) has been shown to be essential for catalytic activity. The catalytic subunit of protein phosphatase 1 specifically dephosphorylates this site, resulting in an inactive kinase that has no intrinsic capacity to rephosphorylate and activate when phosphatase is removed (Dutil et al., 1994). Based on this, and an analysis of the sequence around this site, it has been hypothesized that fransphosphorylation of Thr is catalyzed by an unidentified proline-directed protein... 

Another important family of kinases for drug discovery is the mitogen-activated protein kinases (MAPKs). These are proline-directed serine/threonine kinases that activate their substrates by dual-phosphorylation. MAPK enzymes are activated by a variety of signals including growth factors and cytokines, discussed in chapter 6. The MAPK family plays a critical role in cell cycle progression. Small molecule inhibitors of MAPK may have utility in the treatment of cancer. 

INK (c-Jun N-terminal kinase) was first identified as the UV-induced activity responsible for phosphorylating, and thereby activating the proto-oncogene c-Jun (5). At the same time, they were found as SAPKs (stress-activated protein kinases), which are proline-directed kinases activated by growth factors and biosynthetic inhibitors such as anisomycin. Common stimuli that activate JNKs include inflammatory cytokines fatty acids and environmental stresses such as UV, osmotic shock, heat... 

Kramer RM, Roberts EF, Um SL, Borsch-Haubold AO, Watson SP, Fisher MJ, and Jakubowski JA (1996). p38 mitogen-activated protein kinase jdiosphorylates cytosolic phospholipase A2 (cPLA2) in thrombin-stimulated platelets. Evidence that proline-directed phosph lation is not required ftn-mobilization of arachidonic acid by cPLA2. J. Biol. Chem. 271,27723-27729. 

Two very similar groups of kinases with apparent molecular masses in the 46-57 kDa and 38 kDa ranges, respectively, were soon discovered in experiments with stressful stimuli such as the application of protein biosynthesis inhibitors or exposure of cells to (unphysiological) ultraviolet (UV) radiation (UVC <280 nm) [38] or lipopolysaccharide [39,40]. These stress-activated protein kinases (SAPK) are also proline-directed kinases that are activated by dual phosphorylation. The phosphorylation motif, however, differs from that of the classical MAPK, and is Thr-Pro-Tyr and Thr-Gly-Tyr, respectively, in the two SAPK groups. 

In summary, MAPK are proline-directed Ser/Thr kinases that are activated by dual phosphorylation of a T-X-Y motif (with the exception of the unusual MAPK ERK-3 and ERK-4 Table 2). Their activation is catalyzed by dual specificity (Thr/Tyr) kinases, the MKK, which, in turn, are activated by MKK kinases (MKKK) that are often triggered by interaction with small GTP-binding proteins. 

MBP occurs on specific threonine residues that are proline-directed that is, the phosphorylated amino acids are immediately followed by proline in the amino acid sequence of the protein. In studies of peptide substrates phosphorylated by MAPK it appears that the sequence Ser/Thr-Pro is a minimum consensus phosphorylation sequence for MAPK. However, the amino acids carboxyl- and amino-terminal to this sequence modify the ability of MAPK to covalently attach phosphate. In particular, the placement of proline at position -2 (relative to the phosphorylated amino acid) increases kinase activity. On the basis of these data, the optimal consensus phosphorylation sequence for MAPK is Pro-X-(Ser/Thr)-Pro (Clark-Lewis et al., 1991). Certain proteins, including caldesmon, are phosphorylated by MAPK on sites that do not match this optimal consensus sequence exactly (Adam and Hathaway, 1993). 

Ceramide-activated protein kinase (CAPK) is a proline-directed membrane-bound kinase capable of mediating the effects of ceramide on Rafl kinase in vitro. Kolesnick and co-workers have subsequently identified the kinase suppressor of Ras (KSR) as CAPK and a novel member of the Ras/Raf/MEK (mitogen-activated protein kinase) signaling pathway. Furthermore, it was suggested that ceramide activation of KSR could mediate the effects of TNFa on Rafl (Xing and Kolesnick, 2001). 

Phosphoamino acids that are part of proteins known to bind metal ions are posttranslational modifications introduced by specific protein kinases (Meggio et al, 1981 Vogel and Biidger, 1982c). The bovine milk protein casein and the hen egg-white protein ovalbumin, as well as possibly the human saliva acidic proline-iich proteins share sequence homology of their phosphorylated sites. Dephosphorylation of such sites by enzymatic phosphatase treatment usually reduces the affinity of such proteins for metal-ion binding (Bennick et al., 1981). Hence it is likely that dianionic phosphoryl moieties are directly involved in the complexation of metal ions. This seems particularly important for the two polyelectrolyte proteins that contain large amounts of phosphoserine residues, phosvitin purified from egg yolk (Ta-borsky, 1974), and the phosphoprotein purified from dentine (Linde et al, 1980). 

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