Phosphorylation at the Nuclear Localization Signal of Ca2+/Calmodulin-dependent Protein Kinase II Blocks Its Nuclear Targeting

Abstract

Translocation of protein kinases with broad substrate specificities between different subcellular compartments by activation of signaling pathways is an established mechanism to direct the activity of these enzymes toward particular substrates. Recently, we identified two isoforms of Ca 2؉ /calmodulin-dependent protein kinase II (CaM kinase II), which are targeted to the nucleus by an alternatively spliced nuclear localization signal (NLS). Here we report that cotransfection with constitutively active mutants of CaM kinase I or CaM kinase IV specifically blocks nuclear targeting of CaM kinase II as a result of phosphorylation of a Ser immediately adjacent to the NLS of CaM kinase II. Both CaM kinase I and CaM kinase IV are able to phosphorylate this Ser residue in vitro, and mutagenesis studies suggest that this phosphorylation is both necessary and sufficient to block nuclear targeting. Furthermore, we provide experimental evidence that introduction of a negatively charged residue at this phosphorylation site reduces binding of the kinase to an NLS receptor in vitro, thus providing a mechanism that may explain the blockade of nuclear targeting that we have observed in situ.Phosphorylation and dephosphorylation reactions control a myriad of signal transduction processes within the cell including cell growth and differentiation, metabolic pathways, and gene expression. The specificity of some kinases mediating these reactions is attained by a strict substrate specificity that limits the action of these dedicated kinases to a single or limited number of potential targets. Other kinases, however, are able to phosphorylate a large number of proteins in vitro, so the in vivo specificity of these kinases must occur through a different mechanism. Examples of these multifunctional or general protein kinases include protein kinase A, protein kinase C, and the Ca 2ϩ /calmodulin-dependent protein kinase (CaM kinase) 1 family consisting of CaM kinase I, CaM kinase II, and CaM kinase IV (reviewed in Refs. 1 and 2). All of these kinases are able to phosphorylate nuclear transcription factors in vitro at sites that either activate or repress gene expression and so all of these kinases have the capacity, at least theoretically, to alter cellular phenotype through changes in protein expression. Over the past decade, the evidence that this actually occurs in vivo is becoming increasingly strong; for example, there is now abundant evidence that protein kinase A mediates activation of the cyclic AMP response element-binding protein through phosphorylation of a key Ser residue (3, 4).Nuclear localization of a kinase is necessary for phosphorylation of nuclear proteins such as transcription factors, although there are examples of transcription factors that are activated in the cytoplasm and then translocate to the nucleus (5, 6). The ability of the catalytic subunit of protein kinase A to be released from cytoplasmic tethering and then passively diffuse into the nucleus, where it can phosphorylate nuclear proteins such as cyclic AMP ...

Cytosolic Targeting Domains of γ and δ Calmodulin-dependent Protein Kinase II

“…Constructs C and D target no differently than GFP alone, whereas C-terminal construct E, which lacks central variable domain sequences, targets like full-length CaMK-II. This finding was somewhat surprising, because there are ␥ and ␦ CaMK-II isozymes that have central variable domain (nuclear)-targeting sequences (10,13,14). Because ␦ targeting does not require alternative exons in the variable domain, the ␦ E CaMK-II targeting shown here represents the "default" targeting pattern for ␦ gene products such as ␦ C CaMK-II, which is the principal ␦ CaMK-II isozyme expressed in embryonic cells (6, 8, 10, 12, 21).…”

“…Properly targeted CaMK-IIs can influence cellular events unlike catalytically identical but mistargeted isozymes (20,21). CaMK-II localization can respond to the activation state of CaMK-II (15,22) or to phosphorylation by other kinases (14). Other than nuclear localization sequences, the targeting domains have not been defined for CaMK-II isozymes, particularly those encoded by the ␥ and ␦ genes.…”

“…ticulum (4,10,(13)(14)(15)(16). CaMK-II holoenzyme is normally dodecameric, and targeting can be influenced by the heterooligomerization of CaMK-II monomers (16 -18).…”

p53 regulation by post-translational modification and nuclear retention in response to diverse stresses

“…A protein of unknown function, spot-1, interacts with NLS I, but the signi®cance of this binding remains to be determined (Elkind et al, 1995). The cdc2/cdk2 phosphorylation site at S315 and the PCAF acetylation site at K320 both lie within NLS I, and it is not di cult to imagine that these modi®cations could a ect association with a nuclear import receptor, especially since the phosphorylation of NLSs has been reported to regulate the import of other proteins such as SWI5 (Jans et al, 1995;Moll et al, 1991), Lamin B2 (Hennekes et al, 1993), v-Jun (Tagawa et al, 1995, and CaM kinase II (Heist et al, 1998). However, since an S315A mutation seems to have no e ect on p53 nuclear localization (Liang et al, 1998), it remains to be determined what role these modi®cations may play.…”

Identification and Expression of δ-Isoforms of the Multifunctional Ca 2+ /Calmodulin-Dependent Protein Kinase in Failing and Nonfailing Human Myocardium

“…This apparent antagonism is compensated for by controlling the localization of the holoenzyme whereby the isoform composition and its phosphorylation state regulate translocation to different cellular compartments and control the accessibility to substrates. [11][12][13] As a major regulator of Ca 2ϩ homeostasis, CaMKII is essential for heart function. The importance of this enzyme class for the myocardium is underlined through established cardiac CaMKII targets.…”