Alternative splicing introduces a nuclear localization signal that targets multifunctional CaM kinase to the nucleus

Abstract

Intracellular targeting may enable protein kinases with broad substrate- specificities, such as multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) to achieve a selectivity of action in vivo. We have examined the intracellular targeting of three δ-CaM kinase isoforms. The δ(B)-CaM kinase isoform is targeted to the nucleus in transfected cells while the δ(A)- and δ(C)-CaM kinase isoforms are cytosolic/cytoskeletal. A chimeric construct of α-CaM kinase containing the δ(B)-CaM kinase variable domain is rerouted to the nucleus while the native α-CaM kinase and chimeras of α-CaM kinase which contain the δ(A)- or δ(C)-CaM kinase variable domains are retained in the cytoplasm. Using site-directed mutagenesis, we have defined a nuclear localization signal (NLS) within an 11-amino acid sequence, likely inserted by alternative splicing, in the variable domain of δ(B)-CaM kinase. Isoform-specific nuclear targeting of CaM kinase is probably a key mechanism in the selective regulation of nuclear functions by CaM kinase. CaM kinase is a multimer that can be composed of several isoforms. We find that when cells express two different isoforms of CaM kinase, cellular targeting is determined by the ratio of the isoforms. When an excess of the cytoplasmic isoform of CaM kinase is coexpressed along with the nuclear isoform, both isoforms are localized in the cytoplasm. Conversely an excess of the nuclear isoform can reroute the cytoplasmic isoform to the nucleus. The nuclear isoform likely coassembles with the cytosolic isoform, to form a heteromultimeric holoenzyme which is transported into the nucleus. These experiments demonstrate isoform-specific targeting of CaM kinase and indicate that such targeting can be modified by the expression of multiple isoforms of the enzyme.