Calcium/Calmodulin-dependent Protein Kinase II (CaMKII) Localization Acts in Concert with Substrate Targeting to Create Spatial Restriction for Phosphorylation

Abstract

Ca2؉ /calmodulin-dependent protein kinase II (CaMKII) acts in diverse cell types by phosphorylating proteins with key calcium-dependent functions such as synaptic plasticity, electrical excitability, and neurotransmitter synthesis. CaMKII displays calcium-dependent binding to proteins in vitro and translocation to synaptic sites after glutamatergic activity in neurons. We therefore hypothesized that subcellular targeting of CaMKII can direct its substrate specificity in an activitydependent fashion. Here, we examined whether activitydependent colocalization of CaMKII and its substrates could result in regulation of substrate phosphorylation in cells. We find that substrates localized at cellular membranes required CaMKII translocation to these compartments to achieve effective phosphorylation. Spatial barriers to phosphorylation could be overcome by translocation and anchoring to the substrate itself or to nearby target proteins within the membrane compartment. In contrast, phosphorylation of a cytoplasmic counterpart of the substrate does not require CaMKII translocation or stable protein-protein binding. Cytosolic phosphorylation is more permissive, exhibiting partial calcium-independence. Localization-dependent substrate specificity can also show more graded levels of regulation within signaling microdomains. We find that colocalization of translocated CaMKII and its substrate to lipid rafts in the plasma membrane can modulate the magnitude of phosphorylation. Thus, dynamic regulation of both substrate and kinase localization provides a powerful and nuanced way to regulate CaMKII signal specificity. Ca2ϩ /calmodulin-dependent protein kinase II (CaMKII) 1 is a multifunctional serine/threonine kinase important for a variety of cellular functions including cell division, differentiation, cardiac contraction, and synaptic plasticity (1). CaMKII is required for long term potentiation at CA1 synapses as well as in behavioral learning and memory tasks (2-4). A number of the prominent CaMKII substrates are membrane proteins. CaMKII phosphorylation of the AMPA receptor (AMPAR), ryanodine receptor, and several ion channels regulate their conductance properties (5-7). Thus, CaMKII has a wide repertoire of substrates and associated cellular functions, which raises the question of how CaMKII activity can discriminate among its many substrates. A possible point of regulation could be through the complex subcellular targeting of CaMKII.Several mechanisms of CaMKII subcellular targeting have been characterized as CaMKII splice variants and isoforms can contain targeting sequences such as a nuclear localization sequence and actin binding domains (8, 9). Membrane targeting can be mediated by assembly with proteins such as ␣CaMKII association protein (10). Recently, CaMKII has shown calcium/ calmodulin-dependent interactions with membrane proteins such as the NMDA receptor 2B subunit (NR2B) that can affect its distribution in heterologous cells (11,12). Activity-dependent CaMKII translocation in neurons has also been observed. ...

Differential Modulation of Ca2+/Calmodulin-dependent Protein Kinase II Activity by Regulated Interactions with N-Methyl-D-aspartate Receptor NR2B Subunits and α-Actinin

“…Subunits that are not bound to NR2B presumably remain active to phosphorylate nearby proteins, although it is possible that intersubunit cooperativity allows NR2B to affect the activity of the entire holoenzyme. Consistent with this model, interaction of CaMKII with the high affinity binding site in NR2B was recently shown to enhance phosphorylation of a model substrate engineered onto the same membrane-targeted polypeptide (24). However, dexamethasone treatment of rats decreased the amount of PSD-associated NR2B and increased the amount of PSD-associated CaMKII activity (44).…”

“…For example, some laboratories detect Ca 2ϩ /calmodulin-depend-ent interactions of CaMKII with the NMDAR NR2B subunit that are enhanced by Thr 286 autophosphorylation (21,22), but others detect binding of only [P-T 286 ]CaMKII to NR2B (18). Despite this discrepancy, it is clear that CaMKII activation is important for interactions with NR2B in intact cells (18,21,23,24). In this respect, NR2B remains a strong candidate for mediating the activity-dependent synaptic translocation of CaMKII in neurons.…”

Interleukin-1β-induced Reduction of CD44 Ser-325 Phosphorylation in Human Epidermal Keratinocytes Promotes CD44 Homomeric Complexes, Binding to Ezrin, and Extended, Monocyte-adhesive Hyaluronan Coats

“…The present data indicated that keratinocyte CD44 is also phosphorylated at this site and that the proportion of the phosphorylated form is reduced by the CAMKII inhibitor KN93, like in fibroblasts. The activity of CAMKII could be controlled via phosphorylation or phosphorylation-independent mechanisms, like oxidation, O-GlcNAc modification, and membrane targeting (53)(54)(55). Interestingly, the far end of the cytoplasmic tail of CD44 contains a PDZ binding domain, which binds phosphatases in other transmembrane molecules (Fig.…”

CaMKII inhibition targeted to the sarcoplasmic reticulum inhibits frequency-dependent acceleration of relaxation and Ca2+ current facilitation

“…CaMKII is known to associate with ryanodine receptors (RyRs) [3,4], L-type Ca 2+ channels [5][6][7] and possibly phospholamban (PLB) [8]. This basal localization near targets may be important for efficient and specific regulation of these CaMKII effector proteins.…”