Integrin-linked kinase is responsible for Ca2+-independent myosin diphosphorylation and contraction of vascular smooth muscle

Abstract

Smooth muscle contraction is activated by phosphorylation at Ser-19 of LC20 (the 20 kDa light chains of myosin II) by Ca2+/calmodulin-dependent MLCK (myosin light-chain kinase). Diphosphorylation of LC20 at Ser-19 and Thr-18 is observed in smooth muscle tissues and cultured cells in response to various contractile stimuli, and in pathological circumstances associated with hypercontractility. MLCP (myosin light-chain phosphatase) inhibition can lead to LC20 diphosphorylation and Ca2+-independent contraction, which is not attributable to MLCK. Two kinases have emerged as candidates for Ca2+-independent LC20 diphosphorylation: ILK (integrin-linked kinase) and ZIPK (zipper-interacting protein kinase). Triton X-100-skinned rat caudal arterial smooth muscle was used to investigate the relative importance of ILK and ZIPK in Ca2+-independent, microcystin (phosphatase inhibitor)-induced LC20 diphosphorylation and contraction. Western blotting and in-gel kinase assays revealed that both kinases were retained in this preparation. Ca2+-independent contraction of calmodulin-depleted tissue in response to microcystin was resistant to MLCK inhibitors [AV25 (a 25-amino-acid peptide derived from the autoinhibitory domain of MLCK), ML-7, ML-9 and wortmannin], protein kinase C inhibitor (GF109203X) and Rho-associated kinase inhibitors (Y-27632 and H-1152), but blocked by the non-selective kinase inhibitor staurosporine. ZIPK was inhibited by AV25 (IC50 0.63+/-0.05 microM), whereas ILK was insensitive to AV25 (at concentrations as high as 100 microM). AV25 had no effect on Ca2+-independent, microcystin-induced LC20 mono- or di-phosphorylation, with a modest effect on force. We conclude that direct inhibition of MLCP in the absence of Ca2+ unmasks ILK activity, which phosphorylates LC20 at Ser-19 and Thr-18 to induce contraction. ILK is probably the kinase responsible for myosin diphosphorylation in vascular smooth muscle cells and tissues.

Figure 1. Ca²⁺-independent, microcystin-induced contraction of Triton-skinned rat caudal arterial smooth muscle

Triton-skinned smooth muscle strips contracted in pCa 4.5 and relaxed in pCa 9 solution. Ca²⁺-induced contraction was repeated and relaxation effected again by a return to pCa 9 solution. Only the second Ca²⁺-induced contraction is shown here. Subsequent addition of microcystin (MC; 1 μM) at pCa 9 elicited a contractile response (n=9).

Figure 2. ZIPK and ILK are retained in Triton-skinned rat caudal artery

(A) Western blots probed with anti-ZIPK (left panel) or anti-ILK (right panel): GST–ZIPK (lane 1), chicken gizzard ILK (lane 2), rat bladder (lane 3), rat aorta (lane 4) and rat caudal artery (lane 5). (B) Western blot probed with anti-ZIPK: intact rat caudal artery (one strip; lane 1), Triton-skinned rat caudal artery (one strip; lane 2), chicken gizzard ILK (lane 3) and GST–ZIPK (lane 4). (C) Western blot probed with anti-ILK: GST–ILK (lane 1), chicken gizzard ILK (lane 2), Triton-skinned rat caudal artery (one strip; lane 3) and intact rat caudal artery (one strip; lane 4). Numbers at the right or left of each panel indicate the sizes of molecular-mass markers (in kDa).

Figure 3. In-gel kinase assays

Triton-skinned rat caudal arterial smooth muscle (lanes 1 and 4; two muscle strips), tissue-purified ILK containing a small amount of ZIPK (lanes 2 and 5; 30 μl) and recombinant ZIPK (lanes 3 and 6; 0.125 μg) were subjected to SDS/PAGE in a gel containing purified LC₂₀ (lanes 1–3) or an identical gel without LC₂₀ (lanes 4–6). Following electrophoresis, proteins were renatured in the gels, which were then incubated in the presence of [γ-³²P]ATP and absence of Ca²⁺. Phosphorylated proteins were detected by autoradiography (n=3). Numbers at the right indicate the sizes of molecular-mass markers (in kDa).

Figure 4. Effects of kinase inhibitors on Ca²⁺-independent, microcystin-induced contraction of Triton-skinned, calmodulin-depleted rat caudal artery

(A) Addition of trifluoperazine (TFP; 0.4 mM) at the peak of a Ca²⁺-induced contraction of Triton-skinned strips relaxed the tissue as calmodulin was dissociated [19]. TFP and dissociated calmodulin were removed by several washes in pCa 9 solution. Addition of Ca²⁺ then failed to elicit contraction, confirming the removal of calmodulin, but subsequent addition of microcystin (MC; 1 μM) at pCa 9 activated contraction (n=8). (B–H) Microcystin-induced contraction at pCa 9 was unaffected by the MLCK inhibitors AV25 (100 μM; B) (n=5), ML-7 (100 μM; C) (n=3), ML-9 (300 μM; D) (n=4) or wortmannin (W; 10 μM; E) (n=4), the PKC inhibitor GF109203X (5 μM; F) (n=4) and the ROK inhibitor Y-27632 (10 μM; G) (n=3), but was blocked by the broad spectrum kinase inhibitor staurosporine (STP; 5 μM; H) (n=4).

Figure 5. Inhibition of ZIPK activity by AV25

The activity of purified recombinant ZIPK was assayed with LC₂₀ as substrate in the presence of the indicated concentrations of AV25 as described in the Experimental section. Values are expressed relative to the control (100%=5.4 nmol of P_i/min per mg) in the absence of AV25. Error bars indicate S.E.M. (n=3). The absence of error bars indicates they are smaller than the symbols.

Figure 6. AV25 does not inhibit ILK activity

(A) ILK activity was measured with isolated LC₂₀ as substrate under the indicated conditions, as described in the Experimental section (n=3). Where present, AV25 was at a concentration of 100 μM and staurosporine (STP) at 2 μM; microcystin (MC) was at 1 μM. Values are expressed relative to the control (100%=0.1 nmol of P_i/min per ml) in the absence of kinase inhibitors. Asterisks indicate significant difference from control (P<0.05). (B) Reaction mixtures with LC₂₀ as substrate were analysed by SDS/PAGE and autoradiography. A representative autoradiogram is shown. (C) ILK activity with MLCP (MYPT1) as substrate was assayed by Western blotting with anti-[phospho-Thr-697]-MYPT1 under the indicated conditions. Where present, AV25 was at a concentration of 50 μM.

Figure 7. Effect of AV25 on contraction and LC₂₀ mono- and di-phosphorylation in Triton-skinned rat caudal arterial smooth muscle containing contractile calmodulin

(A) Steady-state force developed in Triton-skinned strips at pCa 9 in the absence or presence of microcystin (MC; 1 μM). Where indicated, tissues were incubated with AV25 (100 μM), staurosporine (STP; 5 μM) or wortmannin (Wort; 10 μM) for 10 min prior to addition of microcystin. (B) Once steady-state force was attained, tissues were quick-frozen for analysis of LC₂₀ phosphorylation by urea/glycerol-PAGE, which separates unphosphorylated, mono- and di-phosphorylated forms of LC₂₀. (C) LC₂₀ bands were quantified by scanning densitometry of gels such as are shown in (B). Different exposure times were used for quantification of these data to ensure that signals lay within the linear range of the relationship between protein amount and signal intensity in each case. Results are expressed as percentages of total LC₂₀ for unphosphorylated (nonP-LC₂₀; open bars), monophosphorylated (P₁-LC₂₀; hatched bars) and diphosphorylated (P₂-LC₂₀; black bars) bands. Phosphorylation stoichiometry was calculated from the following equation: mol of P_i/mol of LC₂₀=(y+2z)/(x+y+z), where x, y and z are the signal intensities of unphosphorylated, mono- and di-phosphorylated LC₂₀ bands respectively. The following phosphorylation stoichiometries were determined: 1.26±0.07 mol of P_i/mol of LC₂₀ (MC alone), 1.28±0.07 mol of P_i/mol of LC₂₀ (MC+AV25) and 1.16±0.14 mol of P_i/mol of LC₂₀ (MC+Wort). n values are indicated below each histogram. Asterisks indicate P<0.001 compared with values at pCa 9 in the presence of microcystin; in all other cases, P>0.3.