Characterization of a calmodulin kinase II inhibitor protein in brain

Abstract

Ca 2؉ ͞calmodulin-dependent protein kinase II (CaM-KII) regulates numerous physiological functions, including neuronal synaptic plasticity through the phosphorylation of ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors. To identify proteins that may interact with and modulate CaM-KII function, a yeast twohybrid screen was performed by using a rat brain cDNA library. This screen identified a unique clone of 1.4 kb, which encoded a 79-aa brain-specific protein that bound the catalytic domain of CaM-KII ␣ and ␤ and potently inhibited kinase activity with an IC 50 Ca2ϩ ͞calmodulin-dependent protein kinase II (CaM-KII) is a widely distributed protein kinase that is particularly abundant in neuronal tissues where it can constitute up to 1-2% of total protein (1). In vitro it can phosphorylate up to 40 proteins, including enzymes, ion channels, transcription factors, etc., and a number of these proteins appear to be physiological substrates. For example, CaM-KII is highly concentrated in the postsynaptic density of glutamatergic synapses where it phosphorylates and potentiates current through the ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptor ion channel (AMPA-Rs) (2-4). This phosphorylation of AMPA-Rs occurs upon induction of long-term potentiation, a model of cellular learning and memory, in region CA1 of hippocampus and is thought to contribute to the postsynaptic current potentiation (2).Oligomeric CaM-KII is comprised of multiple subunits, each composed of an NH 2 -terminal catalytic domain, a central regulatory motif that includes autoinhibitory (AID) and overlapping CaM-binding elements, and a COOH-terminal region involved in subunit assembly (1, 5). The kinase is maintained in an inactive form because of interaction of the AID with the catalytic domain. Binding of Ca 2ϩ ͞CaM conformationally disrupts the AID, allowing access of the catalytic domain to substrates. CaM-KII has the unusual property that upon activation by Ca 2ϩ ͞CaM it exhibits rapid intersubunit autophoshorylation on Thr 286 , generating constitutive kinase activity (6, 7). This constitutive activity prolongs the kinase function beyond the transient elevations of intracellular Ca 2ϩ

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

“…The CaMKII-specific peptide inhibitor antCaNtide is derived from the endogenous CaMKII inhibitor protein CaMKIIN 34 and was made cell permeable by the N-terminal addition of an Antennapedia-derived sequence (antCaNtide: RQI KIW FQN RRM KWK KRP PKL GQI GRS KRV VIE DDR IDD VLK). Calmodulin inhibitors, trifluoperazine (TFP) and N-(6-123 aminohexyl)-5-chloro-1-nafthalene-sulfonamide (W7), PI3-K inhibitor (Ly-294002) and Ras farnesylation inhibitor (lovastatin) were from Sigma.…”

Calcium/Calmodulin-dependent Protein Kinase II Binds to Raf-1 and Modulates Integrin-stimulated ERK Activation

“…The CaMK inhibitor KN93 and the CaM inhibitors trifluoperazine (TFP) and N-(6-aminohexyl)-5-chloro-1-nafthalene-sulfonamide (W7) were purchased from Sigma. The CaMKII specific inhibitor antCaNtide is derived from the endogenous CaMKII inhibitor protein CaMKIIN (26) and was made cell-permeable by N-terminal addition of an Antennapedia-derived sequence (ant-CaNtide: RQIKIWFQNRRMK-WKKRPPKLGQIGRSKRVVIEDDRIDDVLK). The reversed antCaNtide peptide was also used as a control.…”

Inhibitory Phosphorylation of GSK-3 by CaMKII Couples Depolarization to Neuronal Survival

“…Next, we used myr-CaMKIINtide to show that GSK-3 phosphorylation was from CaMKII. Myr-CaMKIINtide is a cell-permeable CaMKII inhibitor derived from CaMKIIN, an endogenous inhibitory protein of CaMKII (58). Myr-CaMKIINtide has been well characterized as a highly selective and potent inhibitor of CaMKII with no effect on CaMKI, CaMKIV, or CaMKK.…”

“…Therefore, the role of CaMKII in neuronal survival supported by depolarization remains ambiguous. To verify specifically the role of CaMKII in survival, we used myr-CaMKIINtide because of its previously validated selectivity and potency (58). As shown in Fig.…”

“…To examine whether the activity of CaMKII␤ is responsible for phosphorylation of GSK-3, CGNs were transfected with CaMKIIN, an endogenous inhibitory protein of CaMKII (58). Expression of CaMKIIN effectively reduced phospho-GSK-3␣/␤ (Ser-21/9) immunostaining in the 25 K condition compared with immunostaining in the vector control (Fig.…”