Dual Mechanism of a Natural CaMKII Inhibitor

Abstract

Ca 2؉ /calmodulin (CaM)-dependent protein kinase II (CaMKII) is a major mediator of cellular Ca 2؉ signaling. Several inhibitors are commonly used to study CaMKII function, but these inhibitors all lack specificity. CaM-KIIN is a natural, specific CaMKII inhibitor protein. CN21 (derived from CaM-KIIN amino acids 43-63) showed full specificity and potency of CaMKII inhibition. CNs completely blocked Ca 2؉ -stimulated and autonomous substrate phosphorylation by CaMKII and autophosphorylation at T305. However, T286 autophosphorylation (the autophosphorylation generating autonomous activity) was only mildly affected. Two mechanisms can explain this unusual differential inhibitor effect. First, CNs inhibited activity by interacting with the CaMKII T-site (and thereby also interfered with NMDA-type glutamate receptor binding to the T-site). Because of this, the CaMKII region surrounding T286 competed with CNs for T-site interaction, whereas other substrates did not. Second, the intersubunit T286 autophosphorylation requires CaM binding both to the "kinase" and the "substrate" subunit. CNs dramatically decreased CaM dissociation, thus facilitating the ability of CaM to make T286 accessible for phosphorylation. Tat-fusion made CN21 cell penetrating, as demonstrated by a strong inhibition of filopodia motility in neurons and insulin secrection from isolated Langerhans' islets. These results reveal the inhibitory mechanism of CaM-KIIN and establish a powerful new tool for dissecting CaMKII function. INTRODUCTIONCa 2ϩ /calmodulin-dependent protein kinase II (CaMKII) is a multifunctional protein kinase best known for its critical role in learning and memory (for review, see Lisman and McIntyre, 2001;Soderling et al., 2001;Hudmon and Schulman, 2002;Lisman et al., 2002). CaMKII is highly expressed in the brain (Erondu and Kennedy, 1985), but at least one of its four isoforms (␣, ␤, ␥, and ␦) has been found in every cell type examined (Tobimatsu and Fujisawa, 1989;Bayer et al., 1999;Tombes et al., 2003). Numerous cellular functions of CaMKII have been described previously, both in and outside the nervous system. These include regulation of various ion channels (Worrell and Frizzell, 1991;Wang and Best, 1992;Roeper et al., 1997;Derkach et al., 1999;Dzhura et al., 2000), gene expression (Nghiem et al., 1994;Ramirez et al., 1997;Meffert et al., 2003), cell cycle/proliferation control (Baitinger et al., 1990;Patel et al., 1999;Matsumoto and Maller, 2002;Illario et al., 2003), apoptotic and excitotoxic cell death (Laabich and Cooper, 2000;Fladmark et al., 2002), cell morphology (Wu and Cline, 1998;Fink et al., 2003), and filopodia motility (Fink et al., 2003). CaMKII also has been implicated in regulation of insulin secretion (for review, see Easom, 1999); however, this conclusion is largely based on experiments using KN inhibitors, which also affect the Ca 2ϩ channels required for secretion (see below).CaMKII forms multimeric holoenzymes (Bennett et al., 1983;Kanaseki et al., 1991;Kolodziej et al., 2000;Morris and Torok, 2001;Hoel...

Synaptic Strength of Individual Spines Correlates with Bound Ca2+–Calmodulin-Dependent Kinase II

“…Specifically, CaMKII could be involved in the organization of a structural link at the PSD (Lisman et al, 2002) and spine cytoskeleton (Narayanan et al, 2006) necessary to stabilize AMPARs at the synapse. Consistent with this notion, recent data directly demonstrated that an inhibitor that interferes with CaMKII binding to NR2B subunit (Vest et al, 2007) significantly suppresses basal synaptic transmission (Sanhueza et al, 2007).…”

Calcium/calmodulin-dependent protein kinase II (CaMKII) phosphorylates Raf-1 at serine 338 and mediates Ras-stimulated Raf-1 activation

“…To evaluate drug interferes competitively with CaMKII/CaM binding, and thus, it is ineffective on the autonomous activity of the kinase. 17 …”

“…Indeed, siRNA only reduced the global cellular CaMKII activity (not shown), while antCaNtide and the CaMKII dominant-negative mutant (K42M, used in subsequent experiments) are recognized as the most specific and potent inhibitors. 17 AntCaNtide pretreatment reduced FCSinduced ERK phosphorylation in a dose-dependent manner. In parallel, ERK phosphorylation induced by KRas V12 was inhibited by antCaNtide treatment.…”

Characterization of a Central Ca2+/Calmodulin-dependent Protein Kinase IIα/β Binding Domain in Densin That Selectively Modulates Glutamate Receptor Subunit Phosphorylation

“…A region of the CaMKII␣ catalytic domain, termed the T-site by Bayer, Schulman, and co-workers, appears to be critical for binding to CaMKIIN, GluN2B, and the CaMKII autoinhibitory domain (23,24,27,28). The role of the T-site in interactions of constitutively active and mCherry-tagged T287D-CaMKII␤ mutant with the densin-IN domain was investigated by mutating Ile-206 and Asp-239 to Lys and Arg, respectively.…”

“…N-tide, a peptide analog containing residues 43-69 of CaMKIIN (26) (termed CN27 by Vest et al (27)), competes with densin-IN for binding CaMKII in vitro. In a crystal structure of CaMKII bound to an N-tide variant (44), amino acid side chains in the N-terminal and central regions of N-tide (underlined in Fig.…”