Back to the future: new techniques show that forgotten phosphorylation sites are present in contractile proteins of the heart whilst intensively studied sites appear to be absent

Abstract

It is well established that several key proteins of the contractile apparatus are phosphoproteins. This includes myosin binding protein C (MyBP-C), troponin T and troponin I. It is also generally accepted that phosphorylation of these proteins alters their functional properties and that this modulation of function through the action of kinases and phosphatases plays a role in tuning the contractile apparatus to physiological demands. The prime example of this is phosphorylation of troponin I and MyBP-C by PKA as part of inotropic and lusitropic responses to b-adrenergic stimulation. What is considerably less certain is the ‘where’ and ‘when’ of these phosphorylations. The sites of contractile protein phosphorylation by kinases such as PKA and PKC have been determined in vitro and the consequences of changes in phosphorylation at these sites have been investigated by site directed mutagenesis and transgenic mouse models. What has not been determined is whether these predominantly in vitro measurements in purified proteins and in rodent models have any relevance to the human heart in vivo. Recently several papers have addressed this question with new techniques and the results have been surprising and somewhat disconcerting. Phosphorylation in human and rat troponin I and troponin T has recently been studied using electrospray ionisation and Fourier transform mass spectrometry (Zabrouskov et al. 2008; Sancho Solis et al. 2008). The great advantage of this method is that the mass of the intact protein can be determined with an accuracy high enough to unambiguously assign the isoform and the nature and number of post-translational modifications, whilst subsequent splitting of the molecular ion can define where in the sequence the modifications are located. When applied to troponin this method reveals the precise patterns of troponin I and troponin T phosphorylation isolated from heart muscle. According to current literature troponin I is phosphorylated in vitro by PKA at Ser22 and 23, by PKC at Ser41, Ser43 and Thr142 and by PAK1 at Ser149 (Noland et al. 1989; Buscemi et al. 2002; Layland et al. 2005) [for consistency we use the numbering of the native human cardiac troponin I sequence in which the N-terminal methionine is removed during post-translational processing (Zabrouskov et al. 2008)]. Measurements of total phosphorylation indicate that 1.6–2.3 mol of Pi are incorporated per mole of troponin I but where is the troponin I phosphorylated (Messer et al. 2009)? One might naturally assume that the troponin I sites phosphorylated in vitro are those phosphorylated in vivo, but under what conditions? Thus far the new mass spectrometry techniques have been applied to whole troponin, purified from healthy unstimulated hearts by antibody affinity methods, to reveal basal phosphorylation patterns (Messer et al. 2007; Sancho Solis et al. 2008). Analysis of human troponin I confirms that Ser22 and 23 are phosphorylated and constitute approximately half of the phosphorylation sites whilst a complementary technique of phosphate affinity SDS–PAGE shows that Ser22 ? Ser23 constitute closer to 2/3 of phosphorylation sites (Messer et al. 2009). Mass spectrometry further reveals the location of these and other prominent sites and somewhat surprisingly shows no sign of phosphorylation at Ser41, Ser43, Thr142 or Ser149. Instead, the remaining 1/2 to 1/3 of S. B. Marston (&) Imperial College London, London SW3 6LY, UK e-mail: s.marston@imperial.ac.uk