Phosphorylation and Dephosphorylation: Protein Kinases A and C

The three subfamilies of PKC are recognized on the basis of sequence similarities and their modes of activation. The conventional PKCs are all activated by phospholipid (in particular phosphatidylserine), DAG, and Ca2 . The novel PKC isoforms require phospholipid and DAG, but not Ca2 . This is explained by the presence of an alternative C2 domain, which does not bind Ca2 . The atypical PKC isoforms respond neither to DAG nor Ca2 and seem not to require phospholipids. They carry an atypical C1 domain and lack the C2 domain. They also have an additional PB1 protein–protein interaction domain. All the PKCs share a common serine/threonine kinase catalytic domain, comprising two highly conserved subdomains, C3 (N-lobe) and C4 (C-lobe). Furthermore, they are all characterized by the presence of a

pseudosubstrate sequence which suppresses kinase activity in the absence of a stimulus. PKCs , , and are widely expressed, whereas expression of the others is largely cell-type specific.

Phosphorylation of substrate by PKC occurs only at serine and threonine residues in the close vicinity of arginine residues situated in the consensus sequence, RxxS/TxRx.49 This is present in many proteins (for a detailed discussion, see Nishikawa et al.50), and as a result the PKCs can be regarded as a family of broad-specificity kinases, with differences in substrate recognition between the subfamilies. Thus, all forms of PKC (with the exception of PKC ) are capable of phosphorylating MARCKS and GAP-43, while ribonucleoprotein A1 (hnR A1) is only efficiently phosphorylated by PKC . The different expression of the various PKC isoforms in the tissues may contribute to particular tissueor cell-specific responses to hormones, growth factors, cytokines, or neurotransmitters.51

Genes coding for pkc2 (conventional), pkc1 and tpa-1 (novel), and pkc3 (atypical) are present in C. elegans.52 Four isotypes, dPKC1, dPKC13, InaC, and a putative dPKC , are present in Drosophila.53 Genetic screening of these

organisms has provided insights into the functioning of PKC, in particular with respect to the role of assembling proteins in the formation of large signalling complexes (see below).

Structural domains and activation of protein kinase C

The C1–C4 regions

The deduced amino acid sequences reveal four reasonably conserved domains,54 C1–C4, having similarities to those present in other signalling proteins.45,55 (Figure 9.9 and Chapter 24). Proceeding from the N-terminus, C1 and C2 constitute the regulatory domains and then C3 and C4 together constitute the catalytic domain characteristic of all kinases (see Chapter 24).

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Signal Transduction

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Phosphorylation and Dephosphorylation: Protein Kinases A and C

FIG 9.10  Activation of conventional protein kinase C occurs in four steps.

Newly synthesized cPKC is processed to render it catalytically competent. This involves three phosphorylations all catalysed by PDK1, most likely at the membrane following generation of DAG and the liberation of Ca2 (1). Membrane binding of PKC (2) allows unfolding. The hydrophobic motif (HM) binds to the N-terminal lobe of PDK1 which causes its activation (3). In return, PDK1 phosphorylates the activation segment of PKC (Table 9.2). This is followed by two autophosphorylations, one on the turn, the other on the hydrophobic motif, which now binds the PKC N-terminal loop (4). At this point, the enzyme is active and said to be fully competent. With depletion of DAG (conversion to phosphatidate) and Ca2 , PKC detaches from the membrane and folds (5). This is caused by the pseudosubstrate ( ) binding in the catalytic cleft and the pseudo-RACK motif with the HM motif. The enzyme remains competent for some time and re-addition of DAG and Ca2 suffice to bring it back to life.

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Signal Transduction

Table 9.2  Phosphorylation sites that render PKC isoforms catalytically competent. The first phosphorylation occurs in the activation segment (T-loop) of the catalytic domain, near the APE sequence. This involves the action of PDK1. The phosphorylations of the turnand hydrophobic motif are autophosphorylations. Note the substitution of the hydrophobic motif serines by a glutamate (E) in PKC and PKC

Adapted from Newton.52

The turn and C-terminal hydrophobic motif

The turn and C-terminal motifs are involved in the binding and the activation of the upstream PDK1 (Figure 9.10). In addition, they control the conformation of the small N-lobe of the PKC and therefore the positioning of the ATPbinding site. Both motifs must be phosphorylated in order to render the kinase fully competent; (exceptions are the atypical PKC and PKC , which have a glutamate in the hydrophobic motif and only require phosphorylation in the turn motif). 

Pseudosubstrate ( ) and pseudoRACK

PKC is kept in an inactive, folded state by (at least) two intramolecular interactions. One involves the segment immediately N-terminal to the C1 domain (residues 19–36 in PKC ) that constitutes the autoinhibitory

pseudosubstrate. The sequence resembles the consensus phosphorylation sites present in target proteins that are phosphorylated by PKC. However, the pseudosubstrate possesses an alanine at the position occupied by serine in the substrates and is consequently not amenable to phosphorylation.61 In the absence of a stimulus, the catalytic domain binds to the pseudosubstrate, causing the enzyme to fold about the hinge linking C2 and C3, and kinase activity is suppressed. The second interaction is a contact between the RACKbinding site (see below), located in the C-terminal region, and a pseudo-RACK motif, located in the C2 domain (Figure 9.10).

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