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 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 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 , 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 -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. PHYSIOLOGY WT ileum SM. Using the speed congenic approach, it was found 2A) after loss of endogenous telokin. In another series of experi- that by the 5th backcross generation, no residual 129-derived ments, the addition of 15 ␮M recombinant S13D telokin, which microsatellite alleles could be detected except in the regions mimics phosphotelokin, to telokin KO ileal SM permeabilized with surrounding MLCK on chromosome 16. Thus, functional differ- Triton X-100, markedly right shifted the pCa force curve, mimick- ences observed in the telokin KO and WT animals are due to the ing the WT curve but, however, did not reach the WT position (Fig. deletion of telokin and not strain-mediated variability in the rest of 8, which is published as supporting information on the PNAS web the genome. The telokin null animals appeared outwardly normal, site). Thus recombinant telokin rescued the pCa force phenotype were fertile, and showed a normal Mendelian inheritance ratio with in telokin-depleted SM, and depletion of telokin in WT SM an average normal litter size of nine pups. recapitulated the KO pCa force phenotype.

Khromov et al. PNAS ͉ February 14, 2006 ͉ vol. 103 ͉ no. 7 ͉ 2441 Downloaded by guest on September 30, 2021 Fig. 3. The leftward shift of the pCa-force curve in telokin KO vs. WT ileal SM is accompanied by an increase in RLC20 phosphorylation.

Maximal force per cross sectional area at pCa 4.5 was not significantly different in KO and WT SM. The leftward shift of Fig. 5. WT compared with KO homogenates of ileal SM exhibit a greater the pCa force curve for the telokin KO ileum compared to the MLCP activity. The time course of RLC20 dephosphorylation induced by mixing WT was correlated with a significant increase in RLC20 phos- phosphorylated SM myosin with ileum SMs homogenates. UREA-PAGE gel phorylation measured at the midpoint (pCa 6.15) of the KO pCa demonstrating different rates of dephosphorylation of phosphorylated SM force curve (paired t test, P Ͻ 0.05; Fig. 3), suggesting either an myosin samples by homogenates from WT and telokin KO ileum SMs. Sum- increase in MLCK or decrease in MLCP activity. Note that mary of the initial rates of dephosphorylation of the prephosphorylated SM myosin by telokin WT and KO homogenates. The rates are expressed as a RLC20 phosphorylation is slightly higher at resting force, pCa 8.0 Ϫ1 Ϫ1 percentage of RLC20 phosphorylation level decrease⅐ min ⅐␮g of homoge- in the telokin KO than in the WT ileum, consistent with a known nate protein (P Ͻ 0.03). basal kinase activity (20), although this phosphorylation did not reach statistical significance. MLCP activity. The content of MYPT1 quantitated from Western Telokin-Deficient Ileum Smooth Muscles Exhibit Reduced 8-Br-cGMP blots and normalized to GAPDH or myosin RCL20 content did not Induced Relaxation of Ca2؉ Force. Telokin is markedly phosphory- significantly differ in WT and telokin KO samples. For example, the lated in SM stimulated by cyclic nucleotides (2, 3). The ability of [MYPT1]:[RLC20] was 0.23 and 0.26 OD in WT and KO and the ϩ 8-Br-cGMP to induce relaxation of Ca2 -induced force was signif- [MYPT1]:[GAPDH] was 0.10 and 0.10 OD in WT and KO, icantly (P Ͻ 0.0005) decreased in ␣-toxin permeabilized telokin KO respectively. Therefore, MLCP protein expression was not altered ileal SM precontracted with pCa 6.3 compared to WT muscles (Fig. by deletion of telokin and, thus, the differences in measured MLCP 4): Magnitudes were 24 Ϯ 3%, n ϭ 5, and 52 Ϯ 1%, n ϭ 7, for KO activity is not due to changes in content of MLCP or MLCK (Fig. and WT ileum muscles, respectively. The sensitivity of SM to 1). MLCP activity was measured in WT and KO whole ileum 8-Br-cGMP did not differ for telokin KO and WT ileal SM, after homogenates (Fig. 5). Initial rates of MLC dephosphorylation were telokin was depleted from WT animals by triton permeabilization, 30% lower in telokin KO mice as compared to WT (15 Ϯ 1% as suggesting that cGMP targets other than telokin are not altered in compared to 19 Ϯ 2% of change in RLC20 phosphorylation⅐ the telokin KO SM (Fig. 4 Inset). minϪ1⅐␮gϪ1 of protein (n ϭ 3, P Ͻ 0.04), respectively. The observed difference in MLCP activity is not due to inhibition of MLCK in Telokin Activates in Vitro and in Vivo MLCP Activity. Whole homog- these experiments because the assay was carried out in the presence enates of ileal SM from telokin WT and KO mice were assayed for of EDTA, thus eliminating MgATP and Ca2ϩ required for MLCK MLCP activity to directly test the hypothesis that telokin activates activity. In vivo MLCP activity was also assayed by using a previously established (21) MLCP activity assay in which the rate of relaxation of Ca2ϩ-activated force measured after removal of Ca2ϩ,bythe addition 10 mM EGTA plus 300 ␮M ML9 to inhibit MLCK activity, is taken as an index of in vivo MLCP activity. The assay is carried out at 10°C to slow the rate of RLC20 dephosphorylation based on the high Q10 value of 5.1 for MLCP (22) and, thus, make it the rate-limiting step. Using this MLCP activity assay, which includes the addition of 10 ␮M 8-Br-cGMP to activate telokin, the slowed rate of 8-Br-cGMP-induced force relaxation in the telokin null ileum SM could be rescued by the addition of S13D recombinant telokin added at the time that inhibition of MLCK activity was initiated (Fig. 6). The t1/2 of relaxation in telokin null ileal SM, 30 Ϯ 0.9 sec (n ϭ 5) was significantly shortened, 25 Ϯ 0.3 sec (n ϭ 5), in the presence of recombinant telokin P Ͻ 0.005. Thus the rate of relaxation reflecting in situ MLCP activity in telokinϪ/Ϫ SM is accelerated by the addition of the phospho-mimic recombinant Fig. 4. 8-Br-cGMP-induced relaxation of Ca2ϩ-induced force was signifi- cantly attenuated by Ͼ50% in ileal SM from telokin KO compared to WT mice. telokin S13D. (Inset) Dose–response curves showing no change in sensitivity for 8-Br-cGMP ؉ 2ϩ induced relaxation upon depletion of telokin, indicating that activation of Ca2 -Sensitized Force in Telokin Null and WT Smooth Muscle. Ca - c-GMP targets are not altered in the telokin KO muscles. sensitized force induced by GTP-␥Sin␣-toxin permeabilized

2442 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0508566103 Khromov et al. Downloaded by guest on September 30, 2021 myosin phosphatase activity, a significant leftward shift of the 2ϩ Ca -force relationship, and an increase in myosin RLC20 phos- phorylation as compared to WT mice. In addition, Ca2ϩ desensi- tization of contractile force by 8-Br-cGMP is reduced by 50% in telokin null intestinal SM and myosin assembled into normal stable thick filaments in the absence of telokin. The Ca2ϩ sensitivity of WT aortic SM was unaffected by the deletion of telokin, likely reflecting its low endogenous telokin content. Taken together, these results, and those of our previous studies (2, 3, 23), support the hypothesis that telokin directly or indirectly increases myosin phosphatase activity and, thereby, contributes to the Ca2ϩ desensitization of force and cyclic nucleotide-induced relaxation in telokin-rich SMs, such as in the gut, bladder, portal vein, resistance vessels, and trachealis. Fig. 6. 8-Br-cGMP-induced relaxation of Ca2ϩ-induced force was signifi- The differences observed with the deletion of telokin are not due cantly accelerated in telokin KO ileal SM upon addition of 10 ␮M S13D to variable genetic backgrounds in the telokin KO mice because the recombinant telokin. At the plateau of the Ca2ϩ-induced force trace, MLCK ͞ ␮ mice were bred to a congenic C57BL 6 background. Furthermore, activity was inhibited by the addition of 300 M ML9 and by removal of the region of the telokin promoter selected for excision and calcium (10 mM EGTA solution, G10) at 10°C. The rate of relaxation is taken as index of in vivo MLCP activity, which is accelerated by the addition of the homologous recombination successfully allowed normal splicing of phospho-mimic recombinant telokin S13D (P Ͻ 0.005). the MYLK gene, as illustrated by the normal expression of MLCK protein and the absence of telokin in ileum SM, which normally expresses high concentrations of telokin (27 ␮M) (23). As expected, 2ϩ telokin KO and WT mice ileal SM contracted with Ca (50% of if telokin increases MLCP activity, the level of RLC20 phosphor- maximal force) did not differ. IC50 ϭ 1.2 Ϯ 0.1 ␮M and 1.3 Ϯ 0.1 ylation should negatively correlate with telokin muscle content. ␮M, n ϭ 5 for WT and KO ileal SM, respectively. Thus endogenous Indeed, resting RLC20 phosphorylation in the phasic guinea pig telokin does not appear to influence the RhoA͞Rho-kinase- portal vein and tonic femoral artery is 10% and 20%, respectively, induced phosphorylation pathway, which leads to inhibition of (20) correlating with the respective high and low telokin expression MYPT1. levels. The 0.3 pCa units leftward shift in the force calcium curve in Morphology of Smooth Muscle from Telokin Null and WT Smooth telokin KO ileal SM results in a 2-fold greater force for a given ϩ Muscle. Electron microscopic evaluation of transverse sections of [Ca2 ] over the physiologically relevant steep portion of the force the longitudinal muscle of the ileum showed a normal array of curve. This shift is unlikely due to adaptive changes in expression myosin filaments throughout the cells in both the telokin KO (Fig. of contractile proteins that occurred secondary to KO of telokin 7) and WT muscles. Longitudinal sections from the carotid artery because the effect could be nearly completely rescued by the also exhibited myosin filaments with no obvious difference due to addition of 15 ␮M S13D telokin. We were unable to discern deletion of telokin. Thus, unlike the in vitro findings demonstrating whether nonphospho- or phospho-telokin was responsible for the that telokin stabilizes myosin filaments (14–16), telokin is not difference in the force calcium curves as both phospho- and necessary for filament formation and stability in the absence of nonphospho-telokin induce relaxation of telokin-depleted muscles telokin. (3), with the S13D phospho-telokin mimic being more potent (10). Indeed, a portion of telokin is in the phosphorylated state in resting Discussion ileum SM, and nonphospho-telokin added to permeabilized mus- The major previously undescribed findings of this study were as cles is labeled with 32P ATP upon stimulation with cGMP (2). follows: (i) that telokin null C57B16 congenic mice survive normally Support for the hypothesis (3) that telokin’s action is through and show no obvious developmental or maturational defects and increased activity of MLCP rather than inhibition of MLCK comes (ii) exhibit normal expression of MLCK but marked decrease in from the following observations. First, we previously showed that telokin had no effect on MLCK-induced thiophosphorylation of RLCs (2) because thiophosphorylated RLCs are not a substrate for MLCP. Second, the presence of telokin did not affect Ca2ϩ-induced force development when MYPT1 was inhibited by microcystin (10). Third, 8-Br-cGMP and recombinant telokin increase the rate and decrease the amplitude of force development upon photolysis of caged ATP (23). Taken together, results support the hypothesis (3) that in vivo, telokin-induced Ca2ϩ desensitization and associated decreases in RLC20 phosphorylation are mediated by acceleration of MLCP activity rather than inhibition of MLCK. NO-induced Ca2ϩ desensitization in resistance arteries (24) is also thought to reflect activation of MLCP. In the present study, we now show directly that MLCP activity is significantly greater in whole homog- enates from WT ileal SM than from telokin KO homogenates, PHYSIOLOGY whereas neither MYPT1 nor MLCK content differed. These findings are further strengthened by the in vivo observations that the rates of relaxation, under conditions where MLCP rates are rate limiting, are significantly slowed in the telokin KO ileal SM but can be rescued by the addition of phospho-mimic S13D recombinant telokin. We have not compared the expected MLCP activity in WT Fig. 7. Electron micrograph illustrating a normal number and distribution of vs. KO muscles based on the pCa-RLC20 phosphorylation mea- myosin filaments in a transverse section of longitudinal ileum SM from a surements with measured MLCP activities because the magnitude telokin null mouse. of the difference in MLCP activity in the KO vs. WT muscle

Khromov et al. PNAS ͉ February 14, 2006 ͉ vol. 103 ͉ no. 7 ͉ 2443 Downloaded by guest on September 30, 2021 homogenates is likely an underestimate, reflecting the assay con- with mechanisms that reduce [Ca2ϩ] and bring about relaxation. ditions of low salt and possible disruption of the physical relation- Although this relationship is not always the case, because NO- ship of the complex of molecules, including telokin partners asso- induced dilations in resistant arteries are not associated with 2ϩ ciated with the RLC20 and possibly the need for native myosin decreases in cytoplasmic [Ca ] (24). We conclude that telokin, a filaments. A similar rationale may account for the inability of target of PKG, through activation of MLCP, plays a significant role recombinant telokin to enhance the in vitro phosphatase activity of in the complex panorama of chemical signal transduction that telokin KO SM homogenates (data not shown) while effectively regulates SM contractility. rescuing in vivo relaxation rates of telokin KO muscles. A well recognized but poorly understood characteristic of tonic Methods arterial SMs is the greater sensitivity of the contractile proteins to Telokin Promoter Deletion. MLCK isoforms (130 kDa and 220 kDa) ϩ Ca2 compared to phasic SMs, such as the portal vein, resistance and telokin are products of a single gene, the MYLK gene (Fig. 1). vessels, bladder, and gut. Although the content of both MLCK and Telokin, a nonenzymatic protein, is an independently regulated MLCP are somewhat less in tonic SMs the ratio of MLCK:MLCP gene embedded within the larger MYLK gene. Telokin expression content are thought to be similar (25). Although the lower con- is mediated by an independent promoter located in an intron centrations could account for the differences in the kinetics of force upstream of exon 29 of the MYLK gene (Fig. 1B) and between the development in these slow and fast SMs, it is unlikely to account for two exons encoding for the calmodulin-binding domain of MLCK. 2ϩ the differences in the Ca -tension relationship. Phasic muscles A 370-bp fragment of telokin is sufficient for maximal cell-specific Ϯ ␮ have a high content of telokin (27 4.6 M in ileum), comparable promoter activity in vitro and to mimic endogenous telokin expres- to the myosin head concentration, whereas the tonic femoral has sion in vivo (34). Within this promoter, an AT-rich region and Ϯ ␮ 4.5-fold less telokin (6 1.7 M) (23). Based on our present adjacent CArG box are critical for promoter activity (19). To findings that the pCa tension curves did not differ in telokin KO and knockdown expression of telokin in vivo and minimize possible 2ϩ WT aortas and that the telokin KO ileum SM had a similar Ca effects on the normal splicing of the MYLK gene, a 30-nt fragment sensitivity as the WT aorta, we propose that telokin and its ability that includes the AT-rich region and CArG box were deleted from to increase MLCP activity accounts for the significant difference in 2ϩ the endogenous telokin gene. The knockout vector for homologous the Ca -force relationship in phasic vs. tonic SM. The result also recombination used Lexicon’s proprietary Lambda KOS system points to a general mechanism of regulation of smooth muscles in (35) (Lexicon Genetics, The Woodlands, TX) (Fig. 1B). The which the contractile properties of different smooth muscles are targeting vector contains 6 kb of 5Ј homology and 3 kb of 3Ј likely to be dictated by the expression levels of distinct subsets of homology. The construct was designed to not significantly alter signaling proteins. 2ϩ exon-intron-spacing (after cre-recombinase-induced excision of the cGMP-induced relaxation of Ca -induced contractions was neomycin phosphotransferase selectable marker gene). Chimeras reduced by 50% in the telokin-deficient ileum under conditions were generated from targeted ES cell lines. The floxed PGKneo where [Ca2ϩ] was held constant, consistent with the increased gene was removed by breeding of the mutant allele through male potency of telokin phosphorylated by PKG (2, 3, 10). The remain- mice expressing Cre recombinase in their germ line. Heterozygotes ing 50% relaxant activity of cGMP is not surprising in view of the were crossed to generate PGKneo-excised homozygotes deficient in multiple targets for active PKG (reviewed in ref. 1). cGMP induces ϩ telokin. Ca2 desensitization of the femoral artery (26) even though telokin content is low and, in this case, is likely signaling to other cGMP Generation of Telokin KO Congenic Lines. A speed congenic approach targets. The ability of 8-Br-cGMP to dramatically reverse GTP-␥S or agonist-induced activation of RhoA͞Rho-kinase mediated Ca2ϩ (36) was used to eliminate the effect of variable genetic back- sensitization of force and (8) demonstrates the antagonistic roles of grounds in the telokin KO mice. The ES cells used for gene ͞ targeting were derived from 129͞SvEvBrd. Chimeric founder males RhoA Rho-kinase and cyclic nucleotide-signaling pathways. How- ͞ ever, PKG-induced Ca2ϩ desensitization can also be shown in were crossed to C57BL 6 albino mice, followed by inbreeding to preparations where the RhoA͞ROK mechanism is inactive (24), confirm the effects of promoter deletion. A male homozygous for the induced mutation was then genotyped to identify an informative and PKG can also increase MLCP activity through interaction ͞ between the leucine zipper motifs of PKG1␣ and MYPT1 (6). microsatellite map for outcross to the C57BL 6 (B6) by using Because GTP-␥S-induced Ca2ϩ sensitization did not change with previously mapped loci (37). Offspring from five successive gener- deletion of telokin, it is unlikely that telokin is inhibiting the ations of backcrossing to the recipient B6 strain were selected for RhoA͞Rho-kinase pathway. Interestingly, in vitro phosphorylation B6 homozygosity at 81 informative microsatellite loci representing of MYPT1 at Ser-695 by PKG͞PKA excludes phosphorylation of a genomewide survey, excluding chromosome Y (which was intro- the adjacent Thr-696 site that is critical for inhibition of MLCP so, duced by a male B6 donor at generation 4). PCR amplification from in this case, cGMP blocks the inhibition of MLCP (27). Which of genomic DNA was performed by using fluorochrome-labeled the above mechanisms account for the remaining cGMP-mediated primers purchased from Research Genetics, (Huntsville, AL) by relaxation in telokin-deficient ileal SM at constant [Ca2ϩ]isnot using a modification of methods in ref. 38. Products from 10–16 loci defined, but it is clear that the absence of telokin strongly abrogates, per animal were pooled after amplification and analyzed individ- by 50%, cGMP-induced relaxation. ually for allele sizes by using an ABI377 automated sequencer The molecular basis for telokin’s ability to lead to an increase in equipped with GENESCAN 2.1 and GENEOTYPER 2.0 software MLCP activity remains to be determined. Telokin can localize to (PerkinElmer Applied Biosystems, Foster City, CA). Once no the S2 region of the myosin head (28) and could either enhance or residual 129-derived microsatellite alleles could be detected, mice inhibit the association of other molecules that target this region such were inbred for propagation of the congenic strain. Subsequent as MYPT1, MYPT kinase (29), integrin-linked kinase (30), myo- heterozygote animals were continued to be backcrossed to the B6 tonic dystrophy-related kinase (31), PKG (6), ROCK, MLCK, strain as a source for the physiological and biochemical analysis. CPI-17 (32), or M-RIP (33), resulting in modulation of MLCP activity, phosphorylation. Genotyping by Southern Blotting and PCR Analyses. Southern blot- The physiological importance of PKG is demonstrated by the ting and PCR analyses were performed to genotype the targeted ES widespread importance of nitrovasodilators and natriuretic pep- clones and weaned pups. For Southern blot analysis, genomic DNA tides in elevating cGMP, resulting in vasodilation and relaxation of was digested with BamHI and probed with 5Ј and 3Ј external probes SMs and physiological or pathophysiological states. In intact mus- as detailed in Supporting Methods, which is published as supporting cles, Ca2ϩ desensitization by PKG generally operates in parallel information on the PNAS web site.

2444 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0508566103 Khromov et al. Downloaded by guest on September 30, 2021 Tissue Preparation, Force, and RLC20 Phosphorylation Measurements. MLCP Assay. Turkey gizzard myosin was prepared and phosphor- Mice were anesthetized and killed with halothane according to ylated at room temperature in buffer as detailed in Supporting protocols approved by the Animal Care and Use Committee at the Methods. MLCP activity of the homogenates from WT and KO University of Virginia. tissue was determined by measuring the rate of dephosphory- Force measurements on intact, ␣-toxin, ␤-escin, or Triton X-100 lation of the prephosphorylated myosin. Tissues were weighed permeabilized muscles were carried out as described previously and and homogenized at 0°C in extraction buffer (50 mM Tris⅐HCl, detailed in Supporting Methods. pH 7.0͞0.1 mM EGTA), plus 1% protease inhibitor mixture at The pCa-force relationships were obtained in permeabilized SMs a ratio of 1 mg of tissue to 20 ␮l of buffer. Total protein content 2ϩ by cumulative increasing the [Ca ] from pCa 6.9 to pCa 4.5, with was adjusted to 0.25 mg͞ml. At 20°C, the homogenate (1 ␮lof each force normalized to the maximal force developed by the same total protein) was mixed with phosphorylated myosin (120 ␮l), strip at pCa 4.5. Normalized pCa-force values obtained for each and the reaction was terminated at 2,5,15, and 30 min by examined strip were fitted to the Hill equation [F ϭ F ϫ max pipetting 30-␮l aliquots of the mixture into the ice-cold acetone (pCa)n͞(pCa)n ϩ EC , where F and F are steady force at given 50 max (1:5 ratio vol͞vol). UREA-Glycerol PAGE was used to separate pCa and maximal force, respectively] to determine the pCa EC50 values. phospho-, and unphospho-RLC20 (39) and determine the per- Ca2ϩ sensitization and 8-Br-cGMP-induced relaxations were cent of phospho-RLC20 to total RLC20 at different time points. determined at intermediate [Ca2ϩ] (pCa 6.3-pCa 6.5) where force The data were fitted to a decaying exponential with rate constant was Ϸ50% of maximal force and characterized with the magnitudes k and the initial rate determined as the product of k and the of the response normalized to steady force developed before initial RLC20 phosphorylation level. MLCP activity was ex- addition of agents. EC50 values were determined by fitting the pressed as an initial rate of MLCP activity decrease per micro- dependences (magnitude vs. [agent]) to a hyperbola. The t1/2 of the gram of homogenate protein. Samples from KO and WT were time course of force decay induced by 8-Br-cGMP was used as an run on the same gel and compared by paired t test. MYPT index of relaxation kinetics, rather than the rate constant, because content in WT and KO homogenate were determined by West- the time course of force decay could not be fitted to a single ern blotting and densitometry over the linear range of protein exponential. In vivo index of phosphatase activity (t1/2 of force loads with MYPT monoclonal (B.D. Transduction relaxation at 10°C) was measured after inhibition of MLCK as Laboratories, Franklin Lakes, NJ). MYPT content was normal- described in ref. 21. ized to RLC20 and GAPDH content. Recombinant mutant telokin was prepared, purified, and buffer exchanged as described in refs. 3 and 10 and used at a concentration Electronmicroscopy. Preparatory procedures were detailed in Sup- of 10–15 ␮M to rescue the force responses. porting Methods. The ratio of phospho-RLC20:total RLC20 was determined by immunoblotting with a rabbit polyclonal phospho-specific antibody We thank Ms. Jama Coartney for preparation of the illustrations and Mr. (Cell Signaling Technology, Beverly, MA) and an antibody to detect Howard Phipps for his preparation of the manuscript. This work was total RLC20 kindly provided by Kris Kamm (University of Texas supported by National Institutes of Health Program Project Grants 5 P01 Southwestern Medical Center, Dallas). HL48807-11A1 and 5 P01 HL19242-29.

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