Nearly 50 years ago, protein phosphorylation was identiﬁed as a regulatory mechanism for the control of glycogen metabolism. It took many years be- fore its general signiﬁcance came to be appreciated. Protein kinases almost regulate every aspect of cell life from growth to death. They mediate most of the signal transduction in eukaryotic cells by regulating substrate activity via phosphorylation. Protein kinases are among the largest families of genes and have been intensively studied. The human genome encodes 518 protein kinases, accounting for 1.7% of all human genes (Manning et al. 2002). These kinases share a catalytic domain conserved in sequence and structure but also notably different in how their catalysis is regulated. The ATP binding pocket together with less-conserved surrounding pockets has been the focus of inhibitor design. Multiple protein kinases have been implicated in pain sen- sitization (Table 1). Importantly, inhibitors of protein kinases do not affect physiological pain: the normal pain threshold in response to mechanical or thermal stimulus remains unchanged. The serine-threonine kinases consist of most protein kinases, and many of serine-threonine kinases are involved in pain regulation (Table 1). There are also 90 tyrosine kinases in the human
Table 1 A list of protein kinases that are known to be involved in inﬂammatory pain (IP), neuropathic pain (NP), peripheral sensitization (PS), and central sensitization (CS). See Sects. 2.1–2.3 for more details
genome including receptor tyrosine kinases and nonreceptor tyrosine kinases (Manning et al. 2002); several tyrosine kinases are implicated in pain facil- itation. In particular, we will discuss the roles of mitogen-activated protein kinases (MAPKs) in regulating pathological pain (see Sect. 2.3), because there is increasing evidence suggesting that this family of serine/threonine kinases is critical for the induction and maintenance of pain hypersensitivity after injuries (Ji and Woolf 2001).
Serine-Threonine Protein Kinases
Protein Kinase A (PKA) This is one of the most classic kinases. Although protein kinase A (PKA) is typically activated by cyclic AMP (cAMP), cAMP’s actions are not exclusively mediated by PKA. cAMP could activate Epac, a guanine nucleotide exchange factor, leading to the activation of p44/42 MAPK and the
ε-isoform of PKC. PKA has been strongly implicated in peripheral and central sensitization. The adenyl cyclase–cAMP–PKA pathway mediates sensitization
of the peripheral terminals of nociceptors induced by PGE2, a major inﬂam- matory mediator (Aley and Levine 1999). PKA is also required for the second phase nociceptive response to formalin and capsaicin-produced mechanical allodynia, two pain models that are thought to result from central sensitization (Coderre and Yashpal 1994; Sluka and Willis 1997). PKA also contributes to the sensitization of spinal-thalamic projection neurons in the lamina I of the
spinal cord following tissue injury (Willis 2002). These NK-1-expressing pro- jection neurons are essential for injury-induced pain hypersensitivity (Man- tyh et al. 1997). In mice with a null mutation of the neuronal-speciﬁc isoform
of PKA’s type I regulatory subunit (RIβ), a selective deﬁcit is found in the development of inﬂammation and tissue injury-produced pain (Malmberg et al. 1997a).
Protein Kinase C PKC is activated by diacylglycerol (DAG) and Ca2+. There is substantial evidence supporting a role of spinal PKC in regulating pain hy- persensitivity in different pain models (Mao et al. 1992; Coderre 1992; Sluka and Willis 1997). PKC is also required for the sensitization of spinal-thalamic projection neurons in the lamina I following intense C ﬁber stimulation (Willis
2002). There is an increase in spinal cord membrane-bound PKC following nerve injury (Mao et al. 1992). In particular, the γ-isoform of PKC is ex- pressed predominantly in inner lamina II neurons of the dorsal horn; there is reduced neuropathic and inﬂammatory pain but preserved acute nocicep- tive pain in mice lacking PKCγ (Malmberg et al. 1997b). PKCγ expression is further induced in the spinal cord by inﬂammation and nerve injury (Mar- tin et al. 1999). The ε-isoform of PKC, in contrast, is expressed in primary sensory neurons and plays a major role in peripheral sensitization of noci- ceptor terminals in inﬂammatory and neuropathic pain conditions (Khasar et al. 1999).
Calcium/Calmodulin-Dependent Kinase This kinase (CaMK-II) has been inten- sively studied in hippocampal neurons and plays an essential role in synaptic plasticity and learning and memory. The active form of αCaMK-II autophos- phorylated at threonine 286, a critical site for CaMK-II activation, has been
shown to enhance excitatory synaptic transmission in dorsal horn neurons
(Kolaj et al. 1994). The superﬁcial dorsal horn is densely stained with an
anti-αCaMK-II antibody (Fang et al. 2002; Zeitz et al. 2004). Recent stud- ies show that CaMK-II is required for central sensitization and neuropathic pain sensitization (Fang et al. 2002; Garry et al. 2003). Disruption of CaMK- II’s docking to the NMDA receptor may be important for the activation of this receptor and the subsequent sensitization of pain behavior after nerve injury (Garry et al. 2003). However, in mice with a point mutation in the
αCaMK-II gene at threonine 286, inﬂammatory pain and neuropathic pain remain unchanged (Zeitz et al. 2004). The role of another isoform, CaMK- IV, in pain regulation is unclear, although it is expressed in 30% of DRG neurons and implicated in the activation of cAMP-response element binding
protein (CREB), an important transcription factor for many neuronal genes
(Ji et al. 1996).
Protein Kinase G (PKG) Ca2+ inﬂux through the NMDA receptor activates the nitric oxide/cGMP/PKG pathway. Neuronal nitric oxide synthase (NOS) is Ca2+/calmodulin-dependent. Nitric oxide (NO) serves as a diffusible and retro- grade messenger. The major target of NO in the CNS appears is a soluble guany-
lyl cyclase (sGC) leading to the production of cyclic guanosine monophosphate (cGMP) and subsequent activation of PKG (Meller and Gebhart 1993). Nerve injury upregulates NOS in DRG neurons (Zhang et al. 1993). PKG appears to play a role in peripheral and central sensitization (Meller and Gebhart
1993; Sluka and Willis 1997; Aley et al. 1998). PKG-I is expressed in the spinal cord, and mice lacking PKG-I show reduced inﬂammatory hyperalgesia with preservation of acute thermal nociception (Tegeder et al. 2004a). In Aplysia sensory neurons, PKG-I couples to the ERK (extracellular signal-regulated ki- nase) pathway and contributes to axotomy-induced long-term hyperexcitabil- ity (Sung et al. 2004).
Other Kinases The transcription factor nuclear factor NF-κB plays an important role in inﬂammatory responses by inducing the transcription of genes encod- ing many inﬂammatory mediators. NF-κB is normally retained in cytoplasm by IκB inhibitor proteins. The phosphorylation of IκB by its kinase, IKK, results in IκB degradation. IκB degradation enables nuclear translocation of NF-κB for gene transcription. Speciﬁc inhibition of IKK is shown to reduce both in- ﬂammatory and neuropathic pain (Tegeder et al. 2004b). Rho kinase (ROCK) is regarded as a promising drug target for neurological disorders (Mueller et al. 2005). ROCK inhibitors were shown to suppress inﬂammatory and neu-
ropathic pain (Inoue et al. 2004; Tatsumi et al. 2005). Casein kinase 2 (CK2) is a widely expressed protein kinase and involved in central sensitization and inﬂammatory pain (Li et al. 2005). Although cyclin-dependent kinase 5 (Cdk5) is typically involved in cell division, recent evidence also suggests a role of this kinase in regulating neural plasticity in the brain. Intrathecal administra- tion of roscovitine, a Cdk5 inhibitor, attenuates formalin-induced nociceptive response in rats (Wang et al. 2005).
Finally, it is worth discussing phosphatidylinositol 3-kinase (PI3K) and the PI3K pathway. Although PI3K is a lipid kinase that phosphorylates the D-3 position of phosphatidylinositol lipids to produce PI(3,4,5)P3, acting asa mem- brane-embedded second messenger (Toker and Cantley 1997), PI3K behaves like a protein kinase. Another reason to include PI3K is that the downstream protein kinase Akt (protein kinase B) is a serine/threonine kinase and is postulated to mediate most of PI3K’s effects. Activation of Akt is typically used to examine the activation of PI3K pathway (Zhuang et al. 2004). PI3K is a major pathway activated by growth factors; therefore, NGF can strongly activate the PI3K pathway in DRG neurons. PI3K is also activated in DRG
neurons by C ﬁber activator capsaicin due to intracellular Ca2+ increase. PI3K inhibitors prevent heat hyperalgesia by both NGF and capsaicin. The PI3K is especially important for the induction of heat hyperalgesia (Zhuang
et al. 2004).