Phosphorylation and Dephosphorylation: Protein Kinases A and C
Activation of protein kinase C
PKC is synthesized as a non-phosphorylated precursor probably associated with the cytoskeleton. At this stage the catalytic site is accessible to ATP but the enzyme is catalytically inactive.62 Although a number of phosphorylations control its activation state, so far only one ‘upstream’ kinase, PDK1, has been identified. It is thought that membrane association, through binding to DAG (and Ca2 ), both increases the chance of meeting PDK1 and then renders
the catalytic domain accessible to its action.63 In this respect, the role of DAG seems equivalent, though not identical, to that of PI(3)P2 in the activation of PKB (see page 550 and Figure 18.6, page 553).
Binding of PKC to the membrane brings about a conformational change in the catalytic domain that separates the catalytic and pseudosubstrate domains. The hydrophobic motif (HM) next binds to the hydrophobic groove in the N-terminal lobe of PDK1, triggering activation segment phosphorylation. Although the conformation remains suboptimal for catalysis, this phosphorylation permits autophosphorylation of the turn and hydrophobic motifs (Figure 9.10) so rendering full competence. The phosphorylation activity of PKC is now directed towards its substrates.52
Things are different for the atypical PKCs and because they do not bind DAG and are insensitive to Ca2 . The activation process may be initiated by other lipid messengers such as PI(3, 4, 5)P3, or the process may be initiated through interaction with proteins such as the Rho-family GTPase Cdc42 in complex with the polarity protein Par-664 (see Figure 9.8 and Figure 19.5, page 586).
Multiple sources of diacylglycerol and other lipids activate protein kinase C
In view of the requirement for diacylglycerol, the regulation of PKC is clearly linked to the activity of PLC (see Chapter 5).65 The hydrophobicity and small size of DAG enables it to diffuse laterally within membranes very rapidly.
Virtually all ligands, growth factors, hormones, or neurotransmitters promote the production of DAG (and IP3) in one way or another, and this means that PKC is implicated in a large number of cellular responses. Sustained activation of conventional PKC requires the simultaneous presence of elevated levels of DAG and high-frequency Ca2 -spikes.66
Several other lipid second messengers and mediators either potentiate the effect of DAG and Ca2 or activate the PKCs directly (Figure 9.11). In particular, the 3-phosphorylated inositol lipids (PI(3,4)P2 and PI(3,4,5)P3, products of the phosphoinositide 3-kinases (see Chapter 18), activate both the novel ( ,, , and ) and the atypical ( , ) PKCs.67 Unsaturated fatty-acids (particularly arachidonic acid and lysophosphatidic acid) and lysophosphatidylcholine
In test-tube experiments, highly purified PKC
can be activated in vitro by the addition of phospholipids, phorbol ester (or diacylglycerol),
and Ca2 (no requirement for PDK1). Moreover, normal phosphorylation occurs at all three processing sites. Based on the reversibility of
this activation following depletion of Ca2 and diacylglycerol, it is suggested that in the cellular environment PKC returns to a closed conformation binding to its pseudosubstrate. It is not known how rapidly and under what conditions phosphatases remove these critical phosphates, although dephosphorylation
has been proposed to accompany the
phorbol ester-mediated down-regulation of PKC activity that follows long exposures times.
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FIG 9.11 Multiple lipid sources, and a protein, to activate protein kinase C. Distinct lipid sources, phospholipases, and kinases activate the different isoforms of PKC. Uniquely, the atypical PKC can also be activated by the GTPase Cdc42 in the presence of Par-6.
also enhance the activity of PKC, potentiating the effects of DAG at resting Ca2 concentrations.68 Conversely, the breakdown products of sphingolipids (sphingosine and lysosphingolipids) inhibit the conventional PKCs, most likely by masking their interaction with phosphatidyl serine.69
Differential localization of PKC isoforms
The existence of numerous isoforms, but the lack of individual substrate specificities, raises the question of whether the various PKCs might have redundant roles. Immunocytochemical analysis, however, reveals that particular isoforms are present in different cells and subcellular compartments and bind to unique protein complexes. They may have specialized functions. The first indication was provided by a study of inductive signalling in Xenopus laevis embryos. Here, dorsal ectoderm is more competent to develop as neural tissue than the ventral ectoderm. The difference persists when an artificial stimulus (phorbol ester) is applied. PKC is preferentially expressed in dorsal ectoderm, whereas PKC is uniformly distributed.70 Over-expression of
PKC in ventral ectoderm annuls the difference, the ventral ectoderm becoming fully competent to form neural tissue.71
Over-expressed isoforms of PKC tend to be diffusely distributed throughout the cytoplasm of fibroblasts, with just a fraction of PKC and PKC attached to the membrane and concentrated in the Golgi apparatus. However, within minutes of adding phorbol ester an extensive redistribution occurs. The andisoforms concentrate at the cell margin. also appears in the endoplasmic reticulum, 2 associates with actin-rich microfilaments of the cytoskeleton
in Golgi, and attaches to the nuclear membrane.72 This differential redistribution effectively dictates access to isoform-specific substrates and ultimately confers functional selectivity and so counteracts the basic lack of substrate selectivity among the various isotypes. The mechanisms that direct the localization are not fully understood.
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Phosphorylation and Dephosphorylation: Protein Kinases A and C
FIG 9.12 RACK1 attaches conventional PKC to the ribosome.
RACK1 binds to the ribosomal 40s subunit and acts to coordinate events arising from conventional PKCs that may have an effect on the translation rate. It also binds -integrins enabling it to localize ribosomes to subcellular compartments.
Adapted from Nilsson et al.76
Different types of PKC-binding proteins
RACKs
Subcellular localization is determined by an array of PKC-binding proteins. Their nomenclature is muddled and they have been called anchors, scaffolds, and receptors. Initially, distinction was made between those that interact with active or inactive PKC and those that are or are not substrates. Proteins that interact exclusively with activated PKC are the RACKs (‘receptors’for activated C kinase).
RACK1 is characterized by seven WD repeats, with a -propeller architecture similar to that of the -subunits of G-proteins (see Figure 4.2, page
84).73 Of these, WD2, 3, and 6 interact with the C-terminal region of PKC (predominantly , , and ). By preventing the interaction with the pseudoRACK motif in the C2 domain, the RACKs could act to maintain the open active conformation of PKC.74 They are thought to anchor activated PKC to particular membrane domains in the vicinity of appropriate substrate proteins. RACK1 links PKC with the intracellular domain of the receptors for interleukin-
5.75 It is also a component of the 40S ribosomal subunit, localized near the head region where mRNA exits the ribosomal particle. RACK1-mediated recruitment of active PKC stimulates translation through phosphorylation of the initiation factor eIF-6 and possibly of mRNA-associated proteins.76 Since RACK1 also interacts with membrane-bound receptors, including integrin - subunits, it promotes the docking of ribosomes at sites where local translation is required, such as focal adhesions (Figure 9.12) and the tips of extending neural growth-cones. Finally, RACK1 links PKC with the JNK protein kinase and in this way enforces expression of cyclinD, giving rise to uncontrolled proliferation of melanocytes (see page 581). RACKII (also known as -COP), interacts with PKC , linking it to the Golgi membrane.77
Substrates that interact with inactive states of PKC
Examples of substrates that interact with PKC in its inactive states are vinculin, talin, MARCKS, - and -adducin, and the annexins. These all play roles in
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Table 9.3 PKC binding proteins
Binding protein/substrate |
Cellular location |
Known functions |
|
|
|
STICKs |
|
|
|
|
|
Vinculin/talin |
focal contacts |
adhesion to extracellular matrix |
|
|
|
Annexins I and II |
– |
– |
|
|
|
MARCKS |
vesicles, plasma membrane |
vesicle trafficking, secretion, cell spreading |
|
|
|
Desmoyokin, AHNAK |
desmosomes/nucleus |
cell-cell binding |
|
|
|
-Adducin |
cortical cytoskeleton |
cell polarity, actin capping |
|
|
|
-Adducin |
cortical cytoskeleton |
interaction of actin with spectrin |
|
|
|
STICK72 and gravin |
plasma membrane |
– |
|
|
|
STICK34 |
cytoskeleton, caveolae |
– |
|
|
|
GAP43 (B-50) |
membrane growth cone |
neurite outgrowth, neuro transmitter release |
|
|
|
P47phox |
neutrophil cytoplasm |
activation NADPH oxidase |
|
|
|
AKAP79 |
synaptic densities |
neurotransmitter release |
|
|
|
PAR-3 |
CNS |
role in cell polarity |
|
|
|
PICKs |
|
|
|
|
|
InaD (Drosophila) |
photoreceptor |
connects PKC with PLC |
|
|
|
ASIP |
tight junction epithelial cells |
– |
|
|
|
PICK1 |
perinuclear |
– |
|
|
|
RACKs |
|
|
|
|
|
RACK1 (binds PKC ) |
interleukin-5 receptor |
– |
|
|
|
-COP (binds PKC ) |
Golgi |
protein traffic |
|
|
|
Adapted from Jaken and Parker.79 |
|
|
linking the actin cytoskeleton with the plasma membrane (Table 9.3). This implies that PKC is a regulator of membrane-cytoskeletal interactions. None of these is selective between different PKC isoforms. In the case of MARCKS and-adducin, phosphorylation disrupts both the interaction with the PKC and with phospholipids, so releasing the protein into the cytosol.78
Proteins that interact with atypical PKCs
Atypical PKC isoforms contain a PB1 domain that interacts with other PB1containing proteins such as the multifunctional cytoplasmic protein p62. This
262