In severe pressure overload-induced
cardiac hypertrophy, a dense, stabilized microtubule network forms that interferes with cardiocyte contraction and microtubule-based transport. This is associated with persistent transcriptional up-regulation of cardiac alpha- and
beta-tubulin and microtubule-stabilizing
microtubule-associated protein 4 (
MAP4). There is also extensive microtubule decoration by
MAP4, suggesting greater
MAP4 affinity for microtubules. Because the major determinant of this affinity is site-specific
MAP4 dephosphorylation, we characterized this in hypertrophied myocardium and then assessed the functional significance of each dephosphorylation site found by mimicking it in normal cardiocytes. We first isolated
MAP4 from normal and pressure overload-hypertrophied feline myocardium; volume-overloaded myocardium, which has an equal degree and duration of
hypertrophy but normal functional and cytoskeletal properties, served as a control for any nonspecific growth-related effects. After cloning
cDNA-encoding feline
MAP4 and obtaining its deduced amino acid sequence, we characterized by mass spectrometry any site-specific
MAP4 dephosphorylation. Solely in pressure overload-hypertrophied myocardium, we identified striking
MAP4 dephosphorylation at Ser-472 in the
MAP4 N-terminal projection domain and at Ser-924 and Ser-1056 in the assembly-promoting region of the C-terminal microtubule-binding domain. Site-directed mutagenesis of
MAP4 cDNA was then used to switch each
serine to non-phosphorylatable
alanine. Wild-type and mutated cDNAs were used to construct adenoviruses; microtubule network density, stability, and
MAP4 decoration were assessed in normal cardiocytes following an equivalent level of
MAP4 expression. The Ser-924 --> Ala
MAP4 mutant produced a microtubule phenotype indistinguishable from that seen in pressure overload
hypertrophy, such that Ser-924
MAP4 dephosphorylation during pressure overload
hypertrophy may be central to this cytoskeletal abnormality.