Centrosomal Anchoring of the Protein Kinase CK1δ Mediated by Attachment to the Large, Coiled-coil Scaffolding Protein CG-NAP/AKAP450

doi:10.1016/S0022-2836(02)00857-4

Journal of Molecular Biology

Volume 322, Issue 4, 27 September 2002, Pages 785-797

https://doi.org/10.1016/S0022-2836(02)00857-4 Get rights and content

Abstract

Protein kinase CK1 (formerly termed casein kinase I) is ubiquitous in eukaryotic cells and comprises a family of as many as 14 isoforms (including splice variants) in mammalian cells. Mammalian CK1δ and CK1ε, which are highly related to each other, are enriched at the centrosomes in interphase cells and at the spindle during mitosis. In the present study we have isolated, using the yeast two-hybrid system, a 182 amino acid residue fragment of the centrosomal and golgi N-kinase anchoring protein (CG-NAP, also known as AKAP450), which specifically interacts with CK1δ and CK1ε, but not with other CK1 isoforms. The 182 amino acid residue CG-NAP fragment, or full length CG-NAP, co-immunoprecipitates with CK1δ and CK1ε from mammalian cells. Consistent with this association, endogenous CG-NAP/AKAP450 and CK1δ co-localize in cells. Moreover, when expressed in the presence of CK1δ the 182 amino acid residue CG-NAP fragment adopts the same sub-cellular localization as CK1δ. Strikingly, attachment of the CG-NAP fragment to the plasma membrane is sufficient to re-localize a significant level of CK1δ to the membrane. These findings support a model in which sub-cellular localization of CK1δ/ε molecules at the centrosome is mediated, at least in part, through the action of CG-NAP/AKAP450 and provide a potential mechanism by which the contribution to cell cycle progression by CK1δ/ε may be regulated.

Introduction

The protein kinase CK1 (formerly termed casein kinase I) comprises a family of serine/threonine protein kinases, which are ubiquitous in eukaryotic systems (for review, see Gross & Anderson¹). Mammalian CK1 members comprise three alternatively spliced α isoforms, together with β, γ1, γ2, γ3, δ and ε isoforms. These enzymes share significant similarity within their protein kinase domains, but show considerable variation in the presence, length and primary structure of the C-terminal non-catalytic domain. The list of known substrates of the CK1 family is limited but includes cytoskeletal proteins such as spectrin, myosin, troponin, ankyrin and tau, as well as nuclear proteins such as RNA polymerases I and II, SV40 T antigen, p53 and CREM¹ (Gross & Anderson,¹ and references therein).

The CK1δ and CK1ε isoforms show striking similarity to each other (98% identity within the protein kinase domain and 40% identity within the non-catalytic C-terminal regions),1., 2., 3. suggesting that these two enzymes may play analogous or overlapping roles in mammalian cells. The C-terminal non-catalytic domains undergo auto-phosphorylation in vitro and in cultured cells treated with phosphatase inhibitors, and could potentially regulate CK1δ/ε function in a physiological context.2., 4., 5. Evidence to date suggests that CK1δ and CK1ε are not restricted to any single pathway but are involved in regulating a variety of cellular events including transduction of the Wnt signaling pathway6., 7. and regulation of the stability and nuclear entry of the PERIOD protein in circadian systems.8., 9., 10. The Saccharomyces cerevisiae CK1 isoform HRR25p is involved in vesicular trafficking,¹¹ the DNA damage response,¹² transcription factor regulation¹³ and G2/M progression.¹² Complementation of the slow growth phenotype of hrr25 cells by CK1δ or CK1ε, but not by other mammalian CK1 isoforms, suggests that CK1δ/ε may be homologues of HRR25p and may play roles in mammalian cells analogous to those of the yeast enzyme.2., 14.

Clues about CK1δ/ε function have also come from cellular localization studies. For example, CK1δ localizes to the golgi and microtubule network in interphase cells, consistent with a role in trafficking.15., 16. Moreover, both CK1δ and CK1ε are enriched in the centrosomes (in interphase) and are tightly associated with the spindle during mitosis.15., 16. Strikingly, DNA damage prior to mitosis appears to stimulate association with the spindle and with tubulins¹⁵ consistent with a role in governing mitotic events in response to cellular stress. The link between CK1δ/ε function, centrosomal localization and cell division is strengthened by the finding that low or sub-micromolar concentrations of IC261, a membrane-soluble CK1-specific inhibitor which has significantly enhanced affinity for CK1δ and CK1ε over other CK1 isoforms,¹⁷ delays cells in the G2/M phases of the cell cycle and can give rise to aberrant mitoses in cells lacking functional p53, consistent with a role for CK1δ/ε in late cell cycle progression.¹⁸

It is presently unclear as to how protein kinases CK1δ/ε, which are apparently constitutively active (at least when their activity is measured in cell extracts) can be pivotal to such a wide range of key cellular events. It is also uncertain as to how (or indeed whether) their activities may be regulated in a cellular context or how they might be recruited to, or organized to play specific roles in, these different processes. One highly attractive hypothesis is that different cellular processes are able to selectively and dynamically recruit these CK1 isoforms under appropriate conditions.

In the present study, we have carried out a search for CK1δ-interacting proteins using the yeast two-hybrid system. Our studies have revealed that the large, coiled-coil centrosomal anchoring protein, CG-NAP,¹⁹ also known as AKAP450,²⁰ contains a domain which interacts specifically with CK1δ and CK1ε, but not with other mammalian CK1 isoforms, consistent with the idea that CG-NAP/AKAP450, which has an established role in anchoring a range of signalling molecules to the centrosome and golgi, also anchors CK1δ/ε to these organelles. This association also provides a potentially striking mechanism by which CK1δ/ε activity may be targeted towards the regulation of molecular events occurring within these organelles, perhaps in coordination with other important signalling molecules.

Section snippets

Isolation of CG-NAP/AKAP450 as a CK1δ interacting protein

In order to search for interacting partners for CK1δ, the yeast two-hybrid system (Clontech) was used to screen a human testis cDNA library (Clontech) fused to the GAL4 activation domain in the pACT2 vector. A cDNA encoding full length CK1δ harbouring a methionine substitution for the essential lysine residue at position 38,¹⁶ was cloned into the yeast expression vector pGBKT7 and used as bait. This catalytically inactive mutant was used initially both as a cautionary procedure on the basis

Discussion

The protein kinase CK1δ, and the highly related CK1ε, are apparently constitutively active signalling enzymes which may play regulatory roles in a variety of cellular events including Wnt signalling, trafficking, nuclear import, DNA damage/repair and cell cycle progression.1., 30. The means by which these CK1 isoforms are regulated in the cellular environment (if at all), and the basis of their involvement in such a wide range of processes in poorly understood. In order to gain insight into the

Cloning of protein kinase CK1δ^m into pGBKT7

A cDNA encoding the wild-type rat protein kinase CK1δ originally isolated by PCR³¹ was subcloned into the NcoI and SalI sites of pGBKT7 to generate (pGBKT7-CK1δ). A catalytically inactive form of CK1δ encoding a lysine to methionine substitution at position 38 (K38M)¹⁶ was subcloned as a 1104 bp ApaI/KpnI fragment into the above vector to generate pGBKT7-CK1δ^m.

Yeast two-hybrid screen and analysis of positive clones

Three different yeast strains PJ69-4A, PJ69-2A (both mating type a) and Y187 (mating type α) were used in the yeast two-hybrid analysis

Acknowledgements

We are grateful to the Medical Research Council (UK) and the Biomedical Research Centre, University of Dundee for support. D.W.M. is the recipient of a Medical Research Council Senior Fellowship.

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^†: Present address: J. E. Sillibourne, Program in Molecular Medicine, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01605, USA.

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Journal of Molecular Biology

Centrosomal Anchoring of the Protein Kinase CK1δ Mediated by Attachment to the Large, Coiled-coil Scaffolding Protein CG-NAP/AKAP450

Abstract

Introduction

Section snippets

Isolation of CG-NAP/AKAP450 as a CK1δ interacting protein

Discussion

Cloning of protein kinase CK1δm into pGBKT7

Yeast two-hybrid screen and analysis of positive clones

Acknowledgements

Cell. Signal.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Cell

Cell

J. Biol. Chem.

Eur. J. Cell Biol.

Exp. Cell Res.

J. Biol. Chem.

J. Biol. Chem.

Trends Cell. Biol.

Curr. Opin. Cell Biol.

J. Mol. Biol.

Curr. Biol.

Cloning of protein kinase CK1δ^m into pGBKT7