A CaMK cascade activates CRE-mediated transcription in neurons of Caenorhabditis elegans

Abstract

Calcium (Ca2+) signals regulate a diverse set of cellular responses, from proliferation to muscular contraction and neuro-endocrine secretion. The ubiquitous Ca2+ sensor, calmodulin (CaM), translates changes in local intracellular Ca2+ concentrations into changes in enzyme activities. Among its targets, the Ca2+/CaM-dependent protein kinases I and IV (CaMKs) are capable of transducing intraneuronal signals, and these kinases are implicated in neuronal gene regulation that mediates synaptic plasticity in mammals. Recently, the cyclic AMP response element binding protein (CREB) has been proposed as a target for a CaMK cascade involving not only CaMKI or CaMKIV, but also an upstream kinase kinase that is also CaM regulated (CaMKK). Here, we report that all components of this pathway are coexpressed in head neurons of Caenorhabditis elegans. Utilizing a transgenic approach to visualize CREB-dependent transcription in vivo, we show that this CaMK cascade regulates CRE-mediated transcription in a subset of head neurons in living nematodes.

Figure 1

CREB homolog of C. elegans (crh-1). (A) An amino acid sequence alignment of KID and bZIP domains of the CREB family from C. elegans (CRH-1β), human (hCREB), mouse (mCREB), zebra finch (zCREB), Drosophila (DmCREB2-a) and hydra (hvCREB). Shading indicates identical amino acids. The asterisk indicates the putative phosphorylated Ser residue of each CREB family member. (B) Analysis of RT–PCR in N2 and crh-1(tz2) worms. cDNAs were amplified with primers from exons 4 and 6 (left) and 4 and 7 (right), respectively. Exon 7 is deleted in crh-1(tz2) strain. (C) Analysis of CRH-1 phosphorylation in N2 and crh-1(tz2) worms. Worm extracts (10 and 20 μl) were prepared with SDS–PAGE sample buffer and subjected to western blot analysis using anti-phospho-CREB antibody. CBB, Coomassie Brilliant Blue.

Figure 2

Phosphorylation and transcriptional regulation of CRH-1β by C. elegans CaMK cascade. (A) Either wild-type (WT) or Ser29Ala (S29A) mutant of CRH-1β (0.2 μg) was incubated with CMK-1 (22 ng) in either the absence or the presence of CKK-1 (0.5 ng) in a solution (25 μl) containing 30 mM HEPES (pH 7.5), 5 mM MgAc₂, 1 mM DTT, 2 mM CaCl₂, 3 μM CaM and either 100 μM [γ-³²P]ATP or 100 μM cold ATP at 30°C for 10 min as described in Methods. After terminating the reaction by the addition of 5 μl of SDS–PAGE buffer, the sample (30 μl for autoradiography and 5 μl for western blot) was subjected to SDS 10% PAGE followed by either autoradiography or western blotting using anti-phospho-CREB antibody or anti-His-tag antibody. Arrows indicate CRH-1β. (B) COS-7 cells (6-well dishes) were transfected with 0.5 μg of a reporter gene (pFR-5 × GAL4-binding element-Luciferase) and expression plasmid (0.5 μg) carrying GAL-4 DNA-binding domain fused with residues 1–242 of either crh-1β wild-type crh-1β or crh-1β (Ser29Ala) and a combination of expression plasmids (pME18s) carrying either cmk-1 wild type or mutants (0.5 μg) and/or ckk-1 (0.5 μg) as indicated. A cDNA of the catalytic subunit of mammalian PKA (0.5 μg) was also used as a positive control. After incubation for 24 h, the cells were deprived of serum for 6 h and then stimulated with (filled bars) or without (open bars) 1 μM ionomycin for 12 h. Then the luciferase activity of each cell extract was measured as described in Methods. Results represent mean ± SEM of three independent transfections.

Figure 3

Expression patterns of ckk-1, cmk-1 and crh-1 in adult hermaphrodites. (Top) The expression pattern of ckk-1::GFP. Arrowheads indicate the expression in vulval muscles. (Middle) The expression pattern of cmk-1::GFP. Arrowheads indicate the expression in vulval muscles. (Bottom) The localization of crh-1 transcripts. Arrows and arrowheads indicate the expression in head neurons and germ cells in an ovary, respectively.

Figure 4

Monitoring CRE-dependent transcription in living nematodes. (A) GFP fluorescence of a transgenic worm carrying only pCRE::GFP (90 ng/μl), wild-type, Ca²⁺/CaM-independent (1–295) or constitutively active forms of cmk-1 (1–295, Thr179Asp). Arrows indicate the expression seen in neurons when a large amount of pCRE::GFP was injected. The arrowhead indicates the typical asymmetrical expression often seen in pharynx neurons when wild-type cmk-1 was injected. (B) Comparison of the number of fluorescent neurons in transgenic animals carrying only pCRE::GFP (30 ng/μl), wild-type, Ca²⁺/CaM-independent (1–295), unphosphorylated (1–295, Thr179Ala) or constitutively active (1–295, Thr179Asp) form of cmk-1 in wild-type animals. (C) Effect of crh-1 and ckk-1 genes for the enhanced expression induced by the constitutively active (1–295, Thr179Asp) or Ca²⁺-independent (1–295) forms of cmk-1. Error bars equal SEM. The p-value (asterisks, <0.0001) in (B) indicates a significant difference in CRE-GFP inducible activity between the wild-type and various forms of cmk-1. The p-value (asterisks, <0.0001) in (C) indicates a significant difference of fluorescence between N2 and either the crh-1 or the ckk-1 mutant.