CaM kinases

Single protein kinases such as PKA, PKC, and Ca +-calmodulin (CaM)-kinases, which result in the phosphorylation of serine and threonine residues in target proteins, play a very important role in hormone action. The discovery that the EGF receptor contains an intrinsic tyrosine kinase activity that is activated by the binding of the hgand EGF was an important breakthrough. The insuhn and IGF-I receptors also contain intrinsic... 

Much evidence supports this scheme. For example, neuronal depolarisation increases the amount of free synapsin in the cytosol and microinjection of CAM kinase II into the terminals of the squid giant axon or brain synaptosomes increases depolarisation-evoked transmitter release. By contrast, injection of dephosphorylated synapsin I into either the squid giant axon or goldfish Mauthner neurons inhibits transmitter release. 

Figure 4.11 Dephosphorylated synapsin, associated with SSVs, is thought to form a heteromeric complex with CAM kinase II (also partially embedded in the vesicular membrane) and actin filaments. An increase in intracellular Ca + triggers phosphorylation of S3mapsin I which dissociates from the vesicular membrane. This frees the vesicles from the fibrin microfilaments and makes them available for transmitter release at the active zone of the nerve terminal...

Other studies have demonstrated that the skeletal muscle ai peptide can be phosphorylated in T-tubule membranes by a multifunctional Ca " /calmodulin (CaM)-dependent protein kinase [111], Phosphorylation occurs on the i subunit to an extent of 2 mol phosphate/mol subunit and on the /i subunit to an extent of 0.7-1 mol phosphate/mol channel [108,111]. Phosphorylation catalyzed by the CaM-kinase on the ai subunit is additive to that caused by PKA and occurs on distinct sites [111]. So far, however, we have not observed any functional consequences of phosphorylation of the skeletal muscle Ca channels by the CaM-kinase. 

PDE1 is phosphorylated by Ca27calmodulin-dependent protein kinase II (CaM-kinase II), which results in decreased affinity of this enzyme for Ca2+/calmodulin and an increase in the concentration of Ca2+ needed for its activation. PDE1 is also phosphorylated by protein kinase A, which likewise decreases its binding to Ca27calmodulin. 

Lisman, J. The CaM kinase II hypothesis for the storage of syaptic memory. Trends Neurosci. 17 406-412,1994. 

Figure 11.8). In addition, the activated enzyme phosphorylates itself, and thus remains partly active even after the Ca2+ concentration falls and calmodulin is released from the enzyme. In contrast to the CaM kinases, another important target of Ca2+-cahnodulin is the plasma membrane Ca2+-ATPase pump, whose activation drives down the Ca2+ concentration within the cell, helping to terminate the signal. 

How the frequency of oscillating Ca signal is decoded or integrated and incorporated into specific biochemical reactions is not understood. There is evidence that the CAM kinase II (see Chapter 7.4.2) is involved in decoding repetitive Ca signals (De Koninck and Schulmaim, 1998). 

The best characterized substrate of Ca Vcalmodulin is the Ca /calmodulin-depen-dent protein kinase (CaM kinase). CaM kinase has an important function in neuronal signal transduction. The mechanism of Ca Vcalmodulin activation of CaM kinase is described in more detail in Section 7.4, together with regulation of protein kinases. Another substrate of Ca Vcalmodulin is myosin light chain kinase (MLCK), involved in contraction of smooth musculature. 

The signal-mediating function of Ca is performed as a Ca Tcahnodulin complex in many signaling pathways. Ca /catmodulin can bind specifically to effector proteins and modulate their activity. In first place as effector proteins of Ca Tcahnodulin are the /calmodulin protein kinases (CaM kinases) (review Braun and Schuhnan, 1995). The CaM kinases are widespread and are found in practically all cells of mam-... 

A rough categorization of the CaM kinases differentiates between specialized CaM kinases and multifunctional kinases. 

An example of a specialized CaM kinase is myosin light chain kinase (MLCK), the primary fimction of which is to phosphorylate the light chain of myosin and thus to control the contraction of smooth musculature. 

The multifimctional CaM kinases are collectively referred to as CaM kinases of type II, whereby further subtypes a, p, y and 6 are differentiated. The a and P subtypes of CaM kinase II only occur in the brain whereas the other subtypes are also found in other organs. The multifunctional CaM kinases regulate many processes (see Table 7.1) such as glycogen metabolism, activity of transcription factors, microfilament formation, synaptic release of neurotransmitters from storage vesicles, biosynthesis of neurotransmitters and many more. An important cellular function is assigned to CaM kinase II in brain, where it makes up 0.25 % of the total protein. 

From a regulatory point of view, CaM kinase II is of particular interest as it has the characteristic of an enzyme with a built-in memory switch". The memory allows the CaM kinase to conserve a stimulatory signal over a longer period of time and to remain in an activated state, even when the initiating stimulus has died away. 

CaM kinase is regulated by both autophosphorylation and by Ca Vcahnodulin. An N-terminal catalytic domain, a regulatory domain and an association domain can be... 

Fig. 7.13. Primary structure and oligomeric structure of CaM kinase II of type p. a) Linear representation of the functional domain of CaM kinase Up. b) The ohgomeric structure shown is proposed for an octamer of type P, based on electron microscopic investigations (Kanaseki et al., 1991). The iV-terminal catalytic domain is represented as a larger circle, the C-terminal ohgomerization domain by a smaller circle. CaM calmodulin.

Fig. 7.14. Regulation of CaM kinase II. Scheme of regulation of CaM kinase II by Ca Vcalmodu-lin and by autophosphorylation. CaM kinase II is inactive in the unphosphorylated form and in the absence of Ca calmodulin. Binding of Ca Vcalmodulin activates the kinase for phosphorylation of protein substrates. In the process, autophosphorylation takes place at a conserved Thr residue that stabilizes the active state of the enzyme. In this state, significant residual activity is still present after dissociation of Ca Vcalmodulin and the enzyme remains in an active state for a longer time after the Ca signal has died away. The active state is only terminated when the activating phosphate residue is cleaved off by a protein phosphatase.

After dissociation of calmodulin, the phosphorylated enzyme stiU has 20—80 % of the activity of the Ca Vcalmodulin bound form. This ensures that significant activity remains after the Ca /calmodulin signal has died away. In the phosphorylated form, CaM kinase is in an autonomous, Ca Vcalmodulin independent state. This is only terminated when phosphatases cleave off the activating phosphate residue and thus lead the enzyme back into the inactive state. 

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