Smooth muscle of telokin-deficient mice exhibits increased sensitivity to Ca2؉ and decreased cGMP-induced relaxation A. S. Khromov*†, H. Wang*†, N. Choudhury*†, M. McDuffie‡, B. P. Herring§, R. Nakamoto*, G. K. Owens*, A. P. Somlyo*¶, and A. V. Somlyo*ʈ Departments of *Molecular Physiology and Biological Physics and ‡Microbiology and Internal Medicine, University of Virginia, Charlottesville, VA 22908; and §Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, IN 46202 Edited by Clara Franzini-Armstrong, University of Pennsylvania School of Medicine, Philadelphia, PA, and approved December 22, 2005 (received for review September 30, 2005) Cyclic nucleotides can relax smooth muscle without a change in RhoA at Ser-188, preventing its binding to Rho-kinase and other 2؉ 2؉ [Ca ]i, a phenomenon termed Ca desensitization, contributing effectors, and this phosphorylation may also play a significant role to vasodilation, gastrointestinal motility, and airway resistance. in Ca2ϩ desensitization and relaxation of SM (8) under some The physiological importance of telokin, a 17-kDa smooth muscle- conditions (reviewed in ref. 1). -specific protein and target for cyclic nucleotide-induced Ca2؉ de- Telokin is another target for cGMP-induced Ca2ϩ desensiti sensitization, was determined in telokin null mice bred to a zation and is abundantly phosphorylated at Ser-13 in SM in congenic background. Telokin null ileal smooth muscle homoge- response to 8-Br-cGMP or forskolin (3, 9, 10). Depletion of nates compared to wild type exhibited an Ϸ30% decrease in endogenous telokin from permeabilized SM correlates with a myosin light-chain phosphatase (MLCP) activity, which was re- loss of 8-Br-cGMP-induced relaxation, which is rescued by the flected in a significant leftward shift (up to 2-fold at pCa 6.3) of the addition of recombinant telokin and further enhanced by PKG Ca2؉ force relationship accompanied by an increase in myosin (2, 3, 10). Site-specific mutation of Ser-13 to an acidic, Asp -light-chain phosphorylation. No difference in the Ca2؉ force rela- residue to mimic phosphorylation, but not to a nonphosphory tionship occurred in telokin WT and knockout (KO) aortas, pre- latable Ala, enhances the Ca2ϩ-desensitizing effect on force. sumably reflecting the normally Ϸ5-fold lower telokin content in Telokin is a small highly acidic protein (11) whose sequence is -aorta vs. ileum smooth muscle. Ca2؉ desensitization of contractile identical to the COOH terminus of SM MLCK and is indepen force by 8-Br-cGMP was attenuated by 50% in telokin KO intestinal dently expressed in SMs through a promoter located within an smooth muscle. The rate of force relaxation reflecting MLCP activ- intron of the MYLK gene (12, 13). Telokin has neither the kinase ity, in the presence of 50 ␮M 8-Br-cGMP, was also significantly domain nor the CaM-binding domain of MLCK and its physi- slowed in telokin KO vs. WT ileum and was rescued by recombinant ological function in vivo has been unclear. In vitro, telokin binds telokin. Normal thick filaments in telokin KO smooth muscles to the S1͞S2 region of unphosphorylated SM myosin (14), indicate that telokin is not required for filament formation or stabilizes unphosphorylated myosin filaments (14–16), modu- stability. Results indicate that a primary role of telokin is to lates the oligomerization of MLCK (17), and inhibits the phos- modulate force through increasing MLCP activity and that this phorylation of myosin by MLCK (14, 17) through competitive effect is further potentiated through phosphorylation by cGMP in inhibition with MLCK for binding to myosin (16). telokin-rich smooth tissues. Because many of the studies on telokin have used permeabilized SMs to manipulate telokin content and this permeabilization may mooth muscle (SM) myosin II ATPase activity and associated give rise to loss of additional diffusible proteins, we have generated Scontraction is activated by actin only when Ser-19 of the myosin telokin null mice to more directly explore the in vivo importance 2ϩ regulatory light chain (RLC20) is phosphorylated. The extent of of telokin in Ca desensitization and relaxation of SM. Because 2ϩ RLC20 phosphorylation is a reflection of the balance between Ca telokin is a SM-specific protein, the effects of the telokin deletion calmodulin-dependent myosin light-chain kinase (MLCK) and is specific to SM. Our results demonstrate the importance of telokin ϩ myosin light-chain phosphatase (MLCP) activities. Although the in regulating the Ca2 sensitivity through activation of MLCP and 2ϩ major mechanism for initiating contraction is the rise in [Ca ]i, in regulating the cGMP-induced relaxation of phasic SM of the force can be further increased or decreased through signaling gastrointestinal, airway, and reproductive tracts. pathways that modulate MLCK and͞or MLCP activities giving rise to processes termed Ca2ϩ sensitization or -desensitization (re- Results viewed in ref. 1). Ca2ϩ sensitization due to inhibition of MLCP Generation of Telokin Null Mice on a Congenic Background. The activity is mediated by an agonist G protein-coupled, Ca2ϩ- 130-kDa and 220-kDa MLCK isoforms and telokin are products of independent process that activates RhoA͞Rho-kinase, phosphor- a single gene, the MYLK gene. Telokin, a protein without enzymatic ylates the myosin regulatory subunit of MLCP (MYPT1), and leads activity, is an independently regulated gene embedded within the to increased force (1). On the other hand, cyclic nucleotide- larger MYLK gene (Fig. 1). Deletion of the a 30-nt fragment activated kinases, in addition to decreasing cytosolic Ca2ϩ, make a containing the AT-rich region and CArG box, which have been significant contribution to dephosphorylation of RLC20 and relax- ation through Ca2ϩ-desensitization processes (2–5) and can reverse G protein-coupled Ca2ϩ sensitization (2, 3). Interaction between Conflict of interest statement: No conflicts declared. the leucine zipper motifs of protein kinase G and MYPT1 leads to This paper was submitted directly (Track II) to the PNAS office. direct stimulation of MLCP (6). A role for this interaction in Ca2ϩ Abbreviations: KO, knockout; MLCK, myosin light-chain kinase; MLCP, myosin light-chain desensitization is supported by the observation that chicken gizzard phosphatase; SM, smooth muscle. MYPT1 lacking the leucine zipper motifs is resistant to 8-Br- †A.S.K., H.W., and N.C. contributed equally to this work. cGMP-induced dephosphorylation of myosin II (7). cGMP-induced ¶Deceased January 14, 2004. ϩ inhibition of Ca2 sensitization mediated by RhoA͞Rho-kinase ʈTo whom correspondence should be addressed. E-mail: [email protected]. signaling may occur through PKG-mediated phosphorylation of © 2006 by The National Academy of Sciences of the USA 2440–2445 ͉ PNAS ͉ February 14, 2006 ͉ vol. 103 ͉ no. 7 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0508566103 Downloaded by guest on September 30, 2021 Fig. 2. Ca2ϩ-force relationship in phasic ileum SM (A) and the tonic aorta (B) from telokin KO and WT mice. The pCa-force relationship is significantly Fig. 1. Generation of the telokin null mouse. (A) The structure and func- shifted to the left in the ␣-toxin permeabilized telokin null ileal SM. Heavy tional domains of 130-kDa and 220-kDa MLCK (modified from ref. 18). (B) permeabilization of WT ileal SM, with 0.5% Triton X-100 resulting in depletion Schematic representation of the MYLK gene and the telokin deletion strategy. of endogenous telokin, also induced a leftward shift of the pCa tension curve The genomic DNA with the MLCK exons is represented by shaded boxes (not of WT animals similar to that observed in the telokin KO muscles. No signifi- all exons are indicated), telokin exons are represented by open boxes, and cant difference was found in the pCa-force relationship between telokin KO transcriptional start sites are indicated by arrows. The start of coding of long and WT abdominal aorta preparations (IC ϭ pCa 6.3 Ϯ 0.1 and 6.4 Ϯ 0.1, and short MLCK and telokin begin, respectively, in exons 1, 15, and 29. The 50 respectively) (B). telokin promoter and the start of telokin exon 1 are in a region that is intronic for MLCK. Plasmid pKOS͞UV1͞29.1 was used as the KO vector with an overall length of Ϸ9 kb. The floxed PGKneo cassette was inserted in such a way that .Increased Ca2؉ Sensitivity of Force in Telokin Null Ileal Smooth Muscle the sequence shown comprising an AT-rich region and critical CArG element of the telokin promoter were deleted during homologous recombination in The pCa force curve was significantly left shifted by 0.3 pCa units, 2ϩ ES cells. (C) Western blot illustrating normal MLCK content in telokin KO and over the physiological range of [Ca ] (Fig. 2A), in ␣-toxin perme- WT ileum and the absence of telokin. abilized ileal SM from the telokin KO animals compared with WT (EC50:KOϭ 6.3 Ϯ 0.05, WT ϭ 6.0 Ϯ 0.06, P Ͻ 0.02 by using the two-tailed t test, df ϭ 9 pairs, t ϭ 2.9). No difference in the Ca2ϩ-force relationship was observed for aorta SM from telokin shown to be critical for promoter activity (19), resulted in a 2ϩ complete loss (Fig. 1C) of telokin protein in mutant animals, with WT and KO animals (Fig. 2B). Note that the Ca sensitivity of the aorta samples is similar to the ileum KO tissue. Permeabilization an occasional animal showing a trace of telokin Ϸ200-fold lower in with 0.5% Triton X-100, a protocol that leads to depletion of the knockout (KO) compared to the WT SM tissues. The content endogenous telokin (10), was used on ileal SM from telokin WT of MLCK protein expression did not differ in the telokin KO and mice. The pCa tension curve was significantly leftward shifted (Fig.